in Polymorphonuclear Neutrophils by Caffeic Acid and Hispidin. Derivatives Isolated from Sword Brake Fern (Pteris ensiformis Burm.). J . Agric. Food Cltem.
Journal ofFa irylakeBota ni ca l Garde n 9(3):
28 ~ 34
(20 10)
Evaluation of Antioxidant Activity and Phytochemical Analysis of Pteris tripartita Sw.- a critically endangered fern from South India Xavier* Ravi Baskaran and Robert Jeyacbandran (Oepanment of Plan t Biology aod Plan t Biotechnology.St. Joseph's College, Tiruchirappalli ,Tamil Nadu- 620 002, India)
Abstract: This study is o utlined to probe the i11 ••iJro antioxidant activi ty o f a criticall y endangered fern, P1eri.1· Jriparlila Sw. The plant extracts were screened fo r their possible antioxidant activity by three comp lementary test systems namely. DPPH free radical scavenging, metal chelati ng assay and phos 1>homolybdcn um method and the resu lts re lated to the total phenol content. The IC,, values of acetone and methanol extracts in DPPH radica l scavenging assay we re obtained to be 77.04 and 11 3.63 11g/ ml respectively. The acetone extract was analyzed by gas chromatography/ mass s pectrometry (GC/MS) and their bioac ti ve compounds were identified. llowever, the IC,,, va lues for tbe s tanda rd BHA, BHT, quercetin a nd rutin we re noted to be 25.58, 4 5.56, 18.79 and 24. 7l 1Jglml respectively. Furthermore. total phenolic
co nt~nts ,
flavon oids. tanni ns a nd vitami n C we re determi ned. Accordingly. the aceto ne extract exhibited highest tota l phenolic
content (67.00:0.28 GAE mg/g). The highest content of total fluvoooids was also observed in ethyl acetate extract (48.24± 1. 28 RE mg/g extract). Owing to having a ntioxida ti ve components. the extracts exhibited satisfying a ntioxidant ac ti vit ies. Our findings demonstrate that the extracts of P. rriparlita possess s ignificant antioxidant activi ties and thus it has great pote ntial as a source for na tural health products. K eywords: i11 vi tro an tiox idaot. DPPH. GC/ MS, natural health products
beginn in g of th e 20 '' century. However, restrict ions o n the
Introduction Oxygen is essential fo r the s urviva l of al l on thi s earth. Durin g th e proces s of oxyge n utiliz ation in norm al physiolog ical and m etabo li c processes approximate ly 5% of
use of th ese c omp o unds a re now be in g impos ed because o f the ir c arc ino geni c ity (Mah davi a nd Sal ukhe, 19 95). C urrentl y, the research o f natura l antiox id an ts like
oxygen gets uni vale ntl y red uced to oxygen deri ved free
a lternative sources of sy nthes is antiox id a nts was emerged
radi cal s ( Yu , 1994; Halli well a nd G utte rid gc ( 1989). However, oxygen -centere d free radi ca ls and o th er reactive
and the exp lo itat ion of th e va rious seco ndary meta bo li tes o f the p lant was high lig hted in rece nt years . T hus, the pheno li c
oxygen species ( ROS), wh ich arc continuou sly, produced in
co mpounds in pa rti cu lar the flavono ids ha ve dra w n
vivo, res ult in ce ll de ath and ti ss ue damage (Ha lli well and G utte ridgc, 1999). Reacti ve oxygen spec ies and ass oc iated free radi c als ha ve bee n implic ated in th e etiology of va riou s human di seases in c ludin g inflammation , m e tab o li c di sorde rs, ce llular aging and atherosclerosis, hea rt di sease, strok e, diabe tes me llitu s, canc e r, mal a ria. rh e umatoid arthr it is and HI V/ A TD S (A iho and Le inon e n, 1999; Odukoya et a/. , 2005). Therefore, radic al scavenge rs g ive promi si ng ind ications of new th erap euti c approaches (Cho e ta/. , 2003). An antioxidant is a compound that inhibits or delays the oxidation of sub strates even if the compound is present in a s ig nificantl y lower co ncentration than is the ox idized sub strate. The scave ngin g of reac ti ve ox ygen species (ROS) is one of the po ss ib le mec han is ms of action of ant ioxid ant co mpound s. T he res t deal s w ith the prevention o f ROS form ation by me tal binding or enzyme
atte nti o n as a potential source ofb ioact ive mol ecul es . Their
inhibition . C hain breaking antioxidants prevent damage by in terfering with the free radica l propa gation casca des (Malko wsk i, 2008).
Synthetic ant iox idant s s uc h as
buty lated hydroxyto lu ene (BHT), butyla ted hydroxyanisole ( BHA ) and prop y l ga llate have been us ed s in ce the
28
fla van nu c leu s struc ture is l inked to t he a nt ioxidant capaci ty. These substances are able to red uce free radica ls like superox ide , peroxyle, a lkoxyle and hydroxy l. The reduct ion occurs by hyd rogen t ransfer react ion to the reactive oxygen species (ROS), (Jovanovic eta/.. 1994 ). Fern s are used in d i fferent tradit ional med ici nal systems of I ndia a nd p lay an imp orta nt ro le in folklo re med ici ne. S ystem atic s urveys of medic inal use of fern ha ve been scarce ly undert ak en wh ic h a re no t pharmaco logically eva luated (Ka ndh asamy ei a/. , 2008: Kau sh ik and Dbim ian ,
1995 ; Di xi t and Bhatt, 1974 : Ma n ic kam and Irudayaraj, 1992). Man y spec ies of ferns and fern a llies a re o f eno rm o us economic utility w ith t he most im portant being the ir medicinal , aesthet ic and food value (Yas uda. 1999). Recen tl y. some ferns ha ve been re ported for ant iox idant activity such as, three S elagin e/la species (Gayat hri et a/.. 2005) , Polypodium leucotomus (Gomba u eta/., 2006) , fi ve Equisetum sp ecies (M i Jovanov ic et a/., 2007), Abacopteris penangiana (Zhao er al.. 2007 ), six folk medic inal fe rn s (Cbang er a/.. 200 7), 3 1 species of fe rns (Ding et a/., 2008).
Evaluation o f Antiox idan t Activi ty a nd Phy tochemical Analysis of Pteris tripartim Sw.- a critically end an ge red fern fr om South India
8/ec lznum orientale Linn (Lai e/ at., 20 I 0), Marsilea quadrifolia (Ripa el a/., 2009), Cheilanthes anceps Sw. (Chowdhary er a!., 2010), Pteris ensiformis Burm (We i et a!. , 2007) and Preris multijlda (Shyur et a/., 2005; Wang et at., 2009). Th e ph yot c he mi ca l po tential o f pte rid ophytes is re lati ve ly une xplo red mo re. a ltho ug h pterid o phytes possess g reat eco no mic po te ntia l due to so m e th erapeuti c va lues (Asakawa, 1982 ; As akawa, 1995 ; C he n et a/., 200 5; Dhim a n, 1998; Gogoi, 2002; Redd y et a/., 200 I ; Sing h et a/., 200L Sing h et a l., 2008; Vas udeva , 1999) . C hemica l invest iga ti o n of more tha n 30 spec ies of the P teridac e ae has been re po rte d (C as till o et a/., 1998). Ph ytoc hemi cal investigatio ns o n the P teris genu s led to iso lation of va ri o us pheno lic comp ounds (Jes uda ss et al., 2 000) . fl avo nol g lycos ides (Salatino and Prado , 199 8; Tana ka eta /. , 1978), ka uran es (Woe rd e n b a g era/., 1 99 6 ) and ptero s insesquite rpenes (Sa ito et al., 1990; Mu rakami eta/. , 198 0). Acc ording t o C ha ndra et al.. (2 008) Pteris trip artita Sw. ( PT) is a c ritica ll y endangered fern from So uth Indi a and it s fr o nd s be in g taken inte rnall y during c hildbirth in Bo uga in ve lle . Thu s. in this study, in vitro ass ays were per form e d to evalua te the a n tiox ida nt ac ti viti es o f crude ex tracts and phy toc he mi ca ls o f P.tripartita were ide ntified us ing GC/ M S technique.
DPPH radical scavenging activity F ree radi ca l scavenging ac t iv ity of PT ext racts was de termined us ing the DPPH method (B lios, 1958) . The a nti oxid a nt capac it ies o f the samp les us ing DPP H as sa y we re compa red w it h tb ose of BH A. BHT, rut in and que rcetin and the blank.
C belating capacity The c he lat ing of ferr o us ions by vario us extrac ts of Ptripartita was estima ted by th e m ethod of Din is er a/., ( 1994 ). Briefl y the extracts (250 ll L) were ad ded to a so luti o n of 2 mmol / L FeC I2 (0 .05 mL) . Th e reaction was initiated by the a ddi t io n of 5 mmol / L fe rroz ine (0.2 mL) a nd the mixture was s haken v igorous ly and left sta nding a t room te mpe rature fo r I 0 mi n . Absorba nce of the so lution was t he n m eas ure d spectroph otometrica lly at 562 nm. Th e chelat ing a cti vity of th e ex tracts wa s eva lua ted usi ng EDTA as st a nd ar d. The resu lts we r e expresse d as m g EDTA equi va lent/ g extrac t.
The a ntioxida nt acti vity of PT sa mples wa s evaluated
Chemicals Fe rri c c hlorid e,
by reduce d pressu re w ith ro tary evaporator and s to red at 4 °C fo r f urther use.
Phosphomolybdenum assay
Materials and Methods l , 1-d i phe n y 1-2 -pi c ry l-h y dr azyl
( DPPH), fer rozin e, ammo nium mo ly bdate.
p e rfo rme d a t solvent bo il ing tempera ture. Afte r t he extrac ti o n, the so lve nt was rem oved from the solute mi xture
vitamin C,
Buty la ted hydroxyani so le (BHA), Buty lated hydroxyto luene ( BHT), querc etin, mtin, sodiu m ph osphate, sodi um ni trite, sodium h ydroxide and AlC l , were purc hased from Himed ia, Merc k or S ig ma. All other reagents used were of ana lytical g rade.
Preparation ofplant extracts The aeri al part of Pteris tripa rlita were co llec ted fro m A lakar hill s of Tamil Nadu and authentification of th e species w as made by Dr.A .Be nnia min , Bota ni ca l S urvey of
by the ph osph omolybdenum me th od ( Prie t o et a/., 1999) . An aliquot o f I 00 11 L o f samp le so lution (in 1 mM d imethy l sul foxid e, DM SO) was comb ined wi th I m L of reagent soluti o n (0 .6 M sul phuric acid , 28 m M so dium phosphate, a nd 4 m M a mmoniu m mo lybdate) in a via l. The vials were cappe d a nd incubated in a wa ter ba th a t 95"C for 90 min. Aft er the sa mp les had coo led to room tempera ture, th e ab sorban ce of the mi xture was measured a t 695 nm against a b lank. Th e res ults re ported (ascorb ic acid equi valen t anti o xida nt ac tiv ity) a re mea n va lues expressed as mg of ascorbic ac id e quiva le nts/g ex tract. Dete rmin ation o f total phenolics a nd tannin s Th e to tal pheno l ic co nte nt was determ ined by Fo lin-
In d ia, Arunac ha l prades h. Its voucher s pec ime n has been
c ioc alte u meth od. Us in g th e same extract th e tannin s were
depos ited in th e Dep artme nt o f Pl ant Bi o logy and Plant
estimated a ft e r treatm e nt w ith polyv in yl po lypyrro lidone
Bi otec hnol ogy o f S t.Joscp h's co lle ge , Tiru cbira pa lli. The sha de dri e d ae rial p art o f Ptripartita ( PT) we re g ro und to a fin e powder and sox hle t extracted sepa rate ly w ith hexane, ace to ne, et hy l acetate, c hlo roform, e th a no l, me th ano l and water. Th e sox hle t procedure co ns isted of g roun d PT sampl es (I 0 g ) pl aced in s ide a thimble loaded into th e soxhle t extractor. The to tal extrac ting time wa s 6 h, a nd th e total amo unt of so lve nt wa s 6 0 ml m a inta ined co ntinu ous ly r e flu xing over th e sa mple. Th e so lve nt assays we re
(P V PP). The am o unt of to tal phe no li cs an d tanninS
were
ex pressed as galli c ac id eq uiva le nts (GA E) in mg p er g ex tract (S iddhu raju a11d Becker, 2003) .
Estim ation of total flavonoids The flavono id con tents of PT ex trac ts were quant ified a nd it acts a s maj o r ant iox id a nts in pl an ts red uc ing ox idati ve s tress. Tb e content was e stima ted as per the method described by Zhi shen er a/., ( 19 99). Initiall y 500j.l-L of all the plant extrac ts we re take n io diffe re nt te st tubes. To
29
Evalua tion of Antioxidant Ac tivity and Phytochemical Analysis of Pteris triparlita Sw.- a criticall y endangered fern from South India
each ex trac ts 2 m l of disti li ed water was added. Then I 50~~ L of NaNO, was added to al l the te st tubes fol lowed by
Results and Discussion
incuba ti on at room tempe rature fo r 6 minu tes . A fte r
DPPH radical scaven ging activity
incuba tion I 50
~L
of AIC I, ( I 0%) was added to al l the test
DPPH assay has been extens ive ly used fo r screening
tubes including th e blank. A ll the tes t tubes were incubated
a ntiox idant ac tivity beca use it can accomm odate many
for 6 minute s at room temp erature. T hen 2 ml ofNaOH was
samples in a short period and is sens it ive enough to detect
added to all the test tubes wh ich we re made up to 5 ml using
act ive ingredients at low concentrations (Sa nchez-Moreno,
d istilled water. The co ntents in all the test tubes we re
2002). DPPH· is a stable nitrogen-cen tered free radica l the
vo rtexed we ll and they we re allowed to stand for I 5 minutes
colo r of w hic h c hanges from v io let to ye ll ow upon redu ction
at room temperature. T he pink colour deve loped due to the
by e ither the process of hydrogen- or e lec tro n- donat ion.
presence of flavo no ids was read spectrophotometrically at
Substances wh ich a re able to perform th is react ion can be
5 10 nm. The amou nt of flavo no ids was ca lc ul ated in rutin
cons idered as antiox idants and therefore radica l scave ngers
equ iva lents.
(Dehpour e t a/.. 2009) . Var ious extracts of P. tripartita
Estimation of Vitamin 'C'
s howed a wide variat ion of IC ,. ranging from 77.04 to
Total vitamin C/asco rb ic acid content was est ima ted in
133 .68i!g/ ml (Table l; Fig. I). Th e highes t an tiox idan t
o rder to determine the antiox idant prope rty of the plant
act ivity was detected in acetone extrac t (77.04 ~tg/ ml). The IC, 0 va lues of other extracts were followed by methanol
under study as ascorb ic acid is a natural a ntiox idant. Its content of PT extracts was determ ined by the me th od formulated by Yen and Chen, ( 1995 ). I 0 mg of sa mple was di sso lved in I 0 m l of l "1.1 meta ph osphoric ac id. Samp le is then filtered through Wba tman No . I filter paper and from this 1 m l of su pernatant was taken and 9 ml dichloro indophenols (Dip ) dye solution was added. Formation of p ink co lor afte r adding of Dip dye so lution shows pre sence of v itamin C. Th e co lor thus formed was read spectropho tomet ri ca ll y at 5 15 nm. Amount of vitamin C was
( 113.63i!g/ ml ), water ( ll6. 2711g/ml), e thyl acetate (1 1 6.82 ~t g / ml),
ethano l ( 117 . 37 J.tg/ m l), ch loroform
(129.87J.tg/ m l) a nd he xa ne extrac t ( 133.6811g / ml ). respective ly. The IC,11 value of standard com po unds were of quercetin (18.79 11g/ m l), ru tin (24. 7 1 J.tg/ ml) , BHA (25.58 J.tgl m l) and BHT (45.56 11g/ m l). T he pheno li c compounds present i n t he ex t r act may contri bu te directly to aotioxidative act ion (Dub e t at.. 1999).
ca lculated in ascorbic acid equivalent (AAE). Water was
Fig. l . DPPH radical sc.lVengingactivity
used as b lank. GC-MS analysis
160 N
The aceto ne ex tract of P.tripartita was analyzed by
140
GC-MS (GC-Thermo ul tra and M S -DSQ II, Ita ly) equipped
e
120
with TR- 5 (The rm o) column. Th e carrier gas was helium.
~
100
The te mperature program was set as fo ll ows: 85 "C hold for 3
a..
~
80
min , raised at 6"C/ min to I 9o•c and 8 · c / min to 250 ·c, a nd
0
bold for i 0 min. T he injector a nd detector temperatures we re se t at 250 and 200 "C, respecti ve ly. The in terface
0
J
co
.,;
"' "'.,.;
60 40 20
0
• Senesl
---..--..--Jjjj
temperature was set at 230"C. Mass spectra were taken at 70 eV. Th e mass range was sca nned from 50 to 600 amu. I d en tifica tion of components The spectrum of the unknown compo nent was compared wi th the s pectrum of the known components stored in N IST a nd Wiley 9 libra ri es. The name and mo lecu lar weight of the components of th e test material s
Plant extracts PTH= hexane extract: PTC= chloroform extract: PTA= ace tone extract: PTEA= ethyl acetate extract: PTE= e-thanol extract: PT M= methanol extract; PTW= water extract. BHA ( Butyl3tcd h yd rox yanisolc). B HT (l3utylatcd hydrox y tol ucnc), querce- tin and ru t in were used as standards.
were ascertained.
Chelating capacity Bi va lent transi tion me tal ions play an important ro le a s
Statistical ana lysis
catalysts of oxidative processes, leading to the format ion o f
A ll exper imen ts were carried out in tripl icate and their results are expressed as mean ± SE (n = 3) . The statisti cal
hydroxyl radi ca ls and hyd ro peroxide d ecomposition reactions v ia Fenton c hemi stry (Halliwell, 1997). ln th e
analyses were perfor m ed by one way ANOVA w ith S PSS
prese nce of chelating agents , the complex format ion is
13.0 so ft wa re a nd re lationships were cons idered to be stati stically s ign ificant w hen P
>
Tme tll'tnt
Vitamin C a lso inhibi ts the conversion of nitrites; chem icals
T able 3 . Phytocompounds ident ified in acetone extr ac t o f P.tripartita by GC-MS.
found in foods and processed meats, into nitrosom ines,
Conclusion
dan gerous cancer ca using compound th at ca n lead to cance r of tb e stomach , bl add er and co lon (John Wort and Smart,
No.
Na me of the compounds
Relt> ntion
Mol ecular
Peak
tim c (min)
weight
area 0/ 0
HexadccatlC
4.71
254
7.24
2
Pcntadecanol
17.68
226
4.97
tannins and v itamin ' C' in P.t ripartila aerial part extracts
3
Octadecuooic ac id. metbyl ester
20.52
295
8.08
Phytochemical analysis using GC-MS
4
a Caryophylknc
21.08
204
4.11
5-Phenyl-l-phcnyl methyl ani no41 1-imidazol[ I ,2c]pyrimidin-7 -one
31.76
31 6
3.68
1,3 Bis(4-tcrt-Buiylphcnyl)2-pbenylpropan-2 -o I
32.57
400
5.43
2004). Table 2 Estimation ofsolvent ex tract recove ry, tota l pbenols.llavo noids,
'l'o hll phenols
tlunmuids
'l'llllnln
VI tamin '
