American Journal of Life Sciences

2013; 1(6): 243-247 Published online November 30, 2013 (http: !!!"s#ien#epublishin$$roup"#om % &%ls) doi: 10"1164' %"&%ls"20130106"11

Extraction of some secondary metabolites & thin layer chromatography from different parts of Centella asiatica L. (UR !
Sanjay R. Biradar, Bhagyashri D. Rachetti
(issue )ulture *&bor&tor+, ,ep&rtment o- .ot&n+, /hri )hh&tr&p&ti /hiv&%i )olle$e 0mer$& 1 413606, ,ist" 0sm&n&b&d, (2/)"3N,34

Email address:
s&n%&+bir&d&r20065redi--m&il"#om (/" 6" .ir&d&r), r&#hettib5$m&il"#om (." ," 6&#hetti)

To cite this article:

/&n%&+ 6" .ir&d&r, .h&$+&shri ," 6&#hetti" 78tr&#tion o- /ome /e#ond&r+ 2et&bolites 9(hin *&+er )hrom&to$r&ph+ -rom ,i--erent P&rts o- Centella Asiatica *" (:6.)" American Journal of Life Sciences. ;ol" 1, No" 6, 2013, pp" 243-247" doi: 10"1164' %"&%ls"20130106"11

Abstract: Centella asiatica (*") :rb" <no!n &s .r&hmi, 3ndi&n Penn+!ort &nd 2&ndoo<&p&rni is & sm&ll herb&#eous &nnu&l pl&nt o- the -&mil+ 4pi&#e&e, 3t is used in tr&dition&l medi#ine -or the tre&tment o- v&rious &ilments 9 &+urvedi# tre&tment -or m&n+ dise&ses" Present stud+ de&ls !ith the =u&lit&tive &nd =u&ntit&tive &n&l+sis o- eth&noli# e8tr&#t o- root, stem 9 le&- o- Centella asiatica (*") :rb" 4 =u&lit&tive &n&l+sis b+ thin l&+er #hrom&to$r&ph+ 9 & =u&ntit&tive &n&l+sis b+ st&nd&rd #hemi#&l proto#ol o- se#ond&r+ met&bolites in the root, stem &nd le&- o- the Centella asiatica *" (:6.) h&ve been studied" :sin$ thin l&+er #hrom&to$r&ph+ ((*)) di--erent #omponents li<e 4l<&loids, /&ponin, >l&vonoids, (erpenoides, Phenol 9 (&nnin &re isol&ted 9 identi-ied" (he 6f v&lues o- the developed spots in the di--erent solvent s+stems &re noted" 3n the =u&ntit&tive &n&l+sis, &l<&loids, s&ponins, terpenoids 9 -l&vonoids &re e8tr&#ted b+ usin$ the st&nd&rd #hemi#&l proto#ol" (hese results m&+ be help-ul -or r&tion&le use o- this pl&nt in the modern s+stem o- he&lth #&re" Keywords: Centella Asiatica L. ?u&ntit&tive 4n&l+sis, (*)

1. ntrod!ction
Pl&nts &re endo!ed !ith v&rious ph+to#hemi#&l mole#ules su#h &s vit&mins, terpenoids, phenoli# &#ids, li$nins, stilbenes, t&nnins, -l&vonoids, =uinones, #oum&rins, &l<&loids, &mines, bet&l&ins, &nd other met&bolites, !hi#h &re ri#h in &ntio8id&nt &#tivit+ @1A @2A" /tudies h&ve sho!n th&t m&n+ o- these &ntio8id&nt #ompounds possess &nti-in-l&mm&tor+, &nti&theros#leroti#, &ntitumor, &ntimut&$eni#, &nti#&r#ino$eni#, &ntib&#teri&l, &nd &ntivir&l &#tivities @3A @4A" (he in$estion o- n&tur&l &ntio8id&nts h&s been &sso#i&ted !ith redu#ed ris<s o- #&n#er, #&rdiov&s#ul&r dise&se, di&betes, &nd other dise&ses &sso#i&ted !ith &$ein$ @BA @6A&nd in re#ent +e&rs, there h&s been & !orld!ide trend to!&rds the use o- the n&tur&l ph+to#hemi#&ls present in berr+ #rops, te&s, herbs, oilseeds, be&ns, -ruits &nd ve$et&bles @7A @CA" Centella asiatica (*inn") :rb&n s+s" s+non+m D+dro#ot+le &si&ti#& *inn" 3t is one othe #hie- herbs -or tre&tin$ s<in problems, to he&l !ounds, -or revit&liEin$ the nerves &nd br&in #ells, hen#e prim&ril+ <no!n &ss & F.r&in -oodF in 3ndi&@10A"(he s#ienti-i# studies h&ve proved & v&riet+ o- bio#hemi#&l #omponents i"e"

se#ond&r+ met&bolites h&ve been -ound in )entell& &si&ti#&" (he #hemi#&l #onstituents o- )entell& pl&nt h&ve & ver+ import&nt role in medi#in&l &nd nutr&#euti#&l &ppli#&tions &nd it is believed due to its biolo$i#&ll+ &#tive #omponents o- triterpenes s&ponins@11A"

Photo Plate 1: Vegetation of Centella asiatica L.

244

/&n%&+ 6" .ir&d&r et al.:

78tr&#tion o- /ome /e#ond&r+ 2et&bolites 9(hin *&+er )hrom&to$r&ph+ -rom ,i--erent P&rts o- Centella Asiatica *" (:6.)

Centella asiatica o- 4pi&#e&e -&mil+ is !idel+ used -or its medi#in&l properties li<e sed&tive, &n&l$esi#, &ntidepressive, &ntimi#robi&l, &ntivir&l &nd immunomodul&tor+@12A" (he triterpenes o- )entell& &re #omposed o- m&n+ #ompounds in#ludin$ &si&ti# &#id, m&de#&ssi# &#id, &si&ti#osside, m&de#&ssoside, br&hmoside, br&hmi# &#id, br&hminoside, th&n<iniside, isoth&n<unisode, #entelloside, m&d&si&ti# &#id, #enti# &#id, &nd #entelli#&#id@13A"

hrs o- e8tr&#tion, e&#h e8tr&#t !&s -iltered throu$h Jh&tm&nKs -ilter p&per no"1 sep&r&tel+" (he -iltr&te !&s ev&por&ted to dr+ness &t room temper&ture 9 store &t B00) in re-ri$er&tor" 2.3.1. $ualitati%e Analysis &y thin "ayer Chro!ato'raphy (1)* 78tr&#t !&s to be$in !ith, #he#<ed b+ (hin *&+er )hrom&to$r&ph+ ((*)) on &n&l+ti#&l pl&tes over sili#& $el" (*) !&s #&rried out to isol&te the prin#iple #omponents th&t !ere present in most e--e#tive e8tr&#ts o- pl&nt" (he di--erent solvent s+stems o- di--erent pol&rities !ere prep&red &nd (*) studies !ere #&rried out to sele#t the solvent s+stem #&p&ble o- sho!in$ better resolution" Method (he &bove prep&red pl&nt e8tr&#ts !ere &pplied on pre-#o&ted (*) pl&tes b+ usin$ #&pill&r+ tubes &nd developed in & (*) #h&mber usin$ suit&ble mobile ph&se" (he developed (*) pl&tes !ere &ir dried &nd observed under ultr& violet li$ht :; &t both 2B4 nm &nd 366 nm" (he+ !ere l&ter spr&+ed !ith di--erent spr&+in$ re&$ents &nd some !ere pl&#ed in hot &ir oven -or 1 min -or the development o- #olor in sep&r&ted b&nds" (he movement othe &n&l+Ee !&s e8pressed b+ its retention -&#tor (6f)" ;&lues !ere #&l#ul&ted -or di--erent s&mple" ,ist&n#e tr&vel b+ solute 6fL

". #aterial and #ethods

2.1. Study Area

Photo plate 2: Collection site - Amrutkund Tq. Basavkalyan, near !a"aras"tra-#arnataka $order.

ist. Bidar

,ist&n#e tr&vel b+ solvent (6f-6etention -&#tor) +etection 4-ter dr+in$ the pl&tes, the+ !ere e8posed to 3odine v&pours b+ pl&#in$ in & #h&mber th&t !&s s&tur&ted !ith iodine v&pours &nd &lso e8posed to di--erent spr&+in$ re&$ents" 4ll pl&tes !ere visu&liEed dire#tl+ &-ter dr+in$ &nd !ith the help o- :; &t 2B4 nm &nd 366 nm in :; (*) vie!er" (he 6f v&lue o- the di--erent pots th&t !ere observed !&s #&l#ul&ted" 2.3.2. $uantitati%e Analysis (he ph+to#hemi#&ls !hi#h &re present in the eth&nol e8tr&#ts o- Centella asiatica !ere determined &nd =u&nti-ied b+ st&nd&rd pro#edures" 2.3.2.1. Al,aloid +eter!ination -sin' .ar&orne (1/03# Method B $ o- the s&mple !&s !ei$hed into & 2B0 ml be&<er &nd 200 ml o- 10M &#eti# &#id in eth&nol !&s &dded &nd #overed &nd &llo!ed to st&nd -or 4 h" (his !&s -iltered &nd the e8tr&#t !&s #on#entr&ted on & !&ter b&th to one-=u&rter o- the ori$in&l volume" )on#entr&ted &mmonium h+dro8ide !&s &dded drop!ise to the e8tr&#t until the pre#ipit&tion !&s #omplete" (he !hole solution !&s &llo!ed to settle &nd the pre#ipit&ted !&s #olle#ted &nd !&shed !ith dilute &mmonium h+dro8ide &nd then -iltered" (he residue is the &l<&loid, !hi#h !&s dried &nd !ei$hed @16A"

/tud+ &re&, 4mrit<und (,ist" .id&r) lies bet!een 17 o 4C G &nd 17o 11GG North l&titudes &nd B60 4BGG minutes &nd 760 4CG e&st lon$itudes, !ith the distri#ts o- NiE&m&b&d &nd 2ed&< in 4ndhr& Pr&desh on the 7&st &nd the distri#ts oN&nded &nd 0sm&n&b&d in 2&h&r&shtr& on the !est" 0n the south lies the distri#t o- Hulb&r$& o- I&rn&t&<&" 2.2. Collection of Plant Materials (he -resh p&rts o- Centella asiatica (*") :rb" !ere #olle#ted in -lo!erin$ period -rom 4mrut<und (=" .&s&v<&l+&n, ,ist" .id&r ne&r 2&h&r&shtr&-I&rn&t&<& border" (he pl&nt m&teri&l !ere properl+ !&shed !ith t&p !&ter &nd then rinsed !ith distilled !&ter, dried in oven &t 600) until pl&nt p&rts be#&me !ell dried -or $rindin$" 4-ter dr+in$, the pl&nt m&teri&ls !ere $round !ell into -ine po!der" 2.3. Preparation of Ethanolic Extract (Root Ste! and "eaf# >or prep&r&tion o- eth&noli# e8tr&#t, & modi-ied method o- 4bdulr&hm&n et"&l (2004) @14A !&s used" (he -resh p&rts o- the pl&nt !ere dried in oven &nd $round to -ine po!der !ith me#h&ni#&l $rinder" (en $r&m o- e&#h pl&nt p&rts !&s then m&#er&ted in 100 ml o- &bsolute eth&nol -or 72 hr" 9 properl+ #overed !ith &luminum -oil 9 l&beled" 4-ter 72

4meri#&n Nourn&l o- *i-e /#ien#es 2013; 1(6): 243-247

24B

2.3.2.2. 1la%onoid +eter!ination &y the Method of 2oha! and 3ocipai4 A&ya5an (1//6# 10 $ o- the pl&nt s&mple !&s e8tr&#ted repe&tedl+ !ith 100 ml o- '0M &=ueous meth&nol &t room temper&ture" (he !hole solution !&s -iltered throu$h !h&tm&n -ilter p&per No 42 (12B mm)" (he -iltr&te !&s l&ter tr&ns-erred into & #ru#ible &nd ev&por&ted into dr+ness over & !&ter b&th &nd !ei$hed to & #onst&nt !ei$ht @17A" 2.3.2.3. Saponin +eter!ination -sin' 7&adoni and 7chu,o (2881# !ethod 10 $ o- s&mples po!der !&s put into & #oni#&l -l&s< &nd B0 ml o- 20M &=ueous eth&nol !ere &dded" (he s&mples !ere he&ted over & hot !&ter b&th -or 4 h !ith #ontinuous stirrin$ &t &bout BBO)" (he mi8ture !&s -iltered &nd the residue re-e8tr&#ted !ith &nother 100 ml 20M eth&nol" (he #ombined e8tr&#ts !ere redu#ed to 40 ml over !&ter b&th &t &bout C0O)" (he #on#entr&te !&s tr&ns-erred into & 2B0 ml sep&r&tin$ -unnel &nd 10 ml o- dieth+l ether !&s &dded &nd sh&<en vi$orousl+" (he &=ueous l&+er !&s re#overed !hile

the ether l&+er !&s dis#&rded" (he puri-i#&tion pro#ess !&s repe&ted" 30 ml o- n-but&nol !&s &dded" (he #ombined n-but&nol e8tr&#ts !ere !&shed t!i#e !ith 10 ml o- BM &=ueous sodium #hloride" (he rem&inin$ solution !&s he&ted in & !&ter b&th" 4-ter ev&por&tion the s&mples !ere dried in the oven to & #onst&nt !ei$ht &nd the /&ponin #ontent !&s #&l#ul&ted &s per#ent&$e @1'A" 2.3.2.6. 9otal 9erpenoides +eter!ination -sin' 1er'uson (1/):# Method 10$ o- pl&nt po!der !ere t&<en sep&r&tel+ &nd so&<ed in &l#ohol -or 24 hours" (hen -iltered, the -iltr&te !&s e8tr&#ted !ith petroleum ether; the ether e8tr&#t !&s tre&ted &s tot&l terpenoids @1CA"

$. Res!lt % Disc!ssion
(*) Pro-ilin$ o- Pl&nt 78tr&#ts in ,i--erent /olvent /+stem )on-irms the Presen#e o- ,iverse Hroup oPh+to#hemi#&ls"

9a&le1: %"ytoc"emical Analysis of different &arts of Centella asiatica L. $y T"in layer c"romatogra&"y. &hemical 'ame Al+aloids Sol(ent System 2:ND40D (17:3) ):2 (1':2) ):H4:2: J (6:2:1:1) .:74 (1:1) )) 6 / * 6 / * 6 / * 6 / * Rf (al!es 0"4', 0"BB 0"64 0"71, 0"7' 0"26, 0"30, 0"4B, 0"B7, 0"'2 0"16, 0"20, 0"26, 0"33, 0"36, 0"4', 0"B1, 0"B7, 0"'2 0"16, 0"30, 0"4B, 0"60, 0"73, 0"'2, 0"'B 0"22 0"10, 0"76 0"0' 0"36, 0"37, 0"'7 0"36,0"3C, 0"'7 0"36, 0"40, 0"46, 0"73, 0"'6 S*ray Reagent 2&+erKs re&$ent

,la(onoid Sa*onins

:; li$ht

3odine v&pours

Ter*enoides

10M D2/04

Note : P P-Pl&nt p&rt", )-#hloro-orm, 2-meth&nol, .-.enEene, 74- 7th+l &#et&te, H4- Hl&#i&l &#et&te, J-!&ter, 6-root, /-stem, *-le&-,

3.1. 9"C for Al,aloid (*) o- root e8tr&#t o- Centella asiatica reve&led the presen#e o- 2 #ompounds h&vin$ 6f v&lues o- 0"4', 0"BB respe#tivel+ !hen & solvent ph&se o- 2eth&nol: ND4 0D (17:3) !&s used" (*) o- stem e8tr&#t o- Centella asiatica reve&led the presen#e o- 2 #ompounds h&vin$ 6f v&lues o0"64 !hen & solvent ph&se o- 2eth&nol: N D 4 0D (17:3) !&s used" (*) o- le&- e8tr&#t o- Centella asiatica reve&led the presen#e o- 2 #ompounds h&vin$ 6f v&lues o- 0"71, 0"7' respe#tivel+ !hen & solvent ph&se o- 2eth&nol: ND4 0D (17:3) !&s used" 3.2. 9"C for 1la%onoid (*) o- root e8tr&#t o- Centella asiatica reve&led the presen#e o- 3 #ompounds h&vin$ 6f v&lues o- 0"26, 0"30, 0"4B, 0"B7, 0"'2 respe#tivel+ !hen & solvent ph&se o)hloro-orm: 2eth&nol (1':2) !&s used" (*) o- stem e8tr&#t o- )entell& &si&ti#& reve&led the presen#e o- 3 #ompounds h&vin$ 6- v&lues o- 0"16, 0"20, 0"26, 0"33, 0"36,

0"4', 0"B1, 0"B7, 0"'2 respe#tivel+ !hen & solvent ph&se o)hloro-orm: 2eth&nol (1':2) !&s used" (*) o- le&e8tr&#t o- Centella asiatica reve&led the presen#e o- B #ompounds h&vin$ 6- v&lues o- 0"16, 0"30, 0"4B, 0"60, 0"73, 0"'2, 0"'B respe#tivel+ !hen & solvent ph&se o)hloro-orm : 2eth&nol (1':2) !&s used"

Photo plate 3: TLC of '. Alkaloids (. )lavonoid, *. Ter&enoides +. Sa&onin of different &arts of Centilla asiatica L.

246

/&n%&+ 6" .ir&d&r et al.:

78tr&#tion o- /ome /e#ond&r+ 2et&bolites 9(hin *&+er )hrom&to$r&ph+ -rom ,i--erent P&rts o- Centella Asiatica *" (:6.)

3.3. 9"C for Saponin (*) o- root e8tr&#t o- )entell& &si&ti#& reve&led the presen#e o- 1 #ompound h&vin$ 6f v&lues o- 0"22 !hen & solvent ph&se o- )hloro-orm: $l&#i&l &#eti# &#id: meth&nol: !&ter (6:2:1:1) !&s used" (*) o- stem e8tr&#t o- Centella asiatica reve&led the presen#e o- 3 #ompounds h&vin$ 6f v&lues o- 0"10, 0"76 respe#tivel+ !hen & solvent ph&se o)hloro-orm: $l&#i&l &#eti# &#id: meth&nol: !&ter (6:2:1:1) !&s used" (*) o- le&- e8tr&#t o- Centella asiatica reve&led the presen#e o- B #ompounds h&vin$ 6f v&lues o- 0"0' !hen & solvent ph&se o- )hloro-orm: $l&#i&l &#eti# &#id: meth&nol: !&ter (6:2:1:1)) !&s used" 3.6. 9"C for 9erpenoids (*) o- root e8tr&#t o- Centella asiatica reve&led the presen#e o- 3 #ompounds h&vin$ 6f v&lues o- 0"36, 0"37, 0"'7 respe#tivel+ !hen & solvent ph&se o- .enEene: 7th+l &#et&te (1:1) !&s used" (*) o- stem e8tr&#t o- )entell& &si&ti#& reve&led the presen#e o- 3 #ompounds h&vin$ 6f v&lues o- 0"36, 0"3C, 0"'7 respe#tivel+ !hen & solvent ph&se o- .enEene: 7th+l &#et&te (1:1) !&s used" (*) o- le&e8tr&#t o- Centella asiatica reve&led the presen#e o- B

#ompounds h&vin$ 6f v&lues o- 0"36, 0"40, 0"46, 0"73, 0"'6 respe#tivel+ !hen & solvent ph&se o- .enEene: 7th+l &#et&te (1:1) !&s used" (*) pro-ilin$ o- &ll e8tr&#ts $ives &n impressive result th&t dire#tin$ to!&rds the presen#e o- number oph+to#hemi#&ls" ;&rious ph+to#hemi#&ls $ives di--erent 6f v&lues in di--erent solvent s+stem" (his v&ri&tion in 6f v&lues o- the ph+to#hemi#&ls provides & ver+ import&nt #lue in underst&ndin$ o- their pol&rit+" >rom the e&rlier report <ne! th&t Centella asiatica !&s tr&dition&ll+ used -or m&n+ dise&ses p&rti#ul&rl+ the pl&nt !&s identi-ied &nd &uthenti#&ted bot&ni#&ll+" (he Pl&nt p&rts (6oot, /tem &nd *e&-) !ere sh&de dried &nd $round to the #o&rse po!der" .&sed upon the prelimin&r+ ph+to#hemi#&l test ?u&ntit&tive determin&tion ph+to#onstituents !ere #&rried out -or the po!dered pl&nt m&teri&l b+ v&rious st&nd&rd methods &nd -ound th&t &l<&loid 0"01$m, 0"1 &nd 0"2 $m in root, stem, le&respe#tivel+, -l&vonoids 0"1$m, 0"3$m &nd 1"4$m in root, stem, le&- respe#tivel+, terpenoids 0"1$m, 0"1$m &nd 0"7$m in root, stem, le&- resp" &nd /&ponin 0"1$m, 0"2$m &nd 0"2$m in root, stem, le&- respe#tivel+"

9a&le 2:- ,uantitative analysis of Centella asiatica L. Al+aloid n gm E0traction. Each 1 gm 0"01 0"1 0"2 0"02B 0"1' 0"44 Al+aloid -/. ,la(onoid. -gm. E0traction Each 12 gm 0"1 0"3 1"4 0"2B 0"B6 3"0 ,la(onoid -/. Ter*enoid -gm. E0traction Each 12 gm 0"1 0"1 0"7 0"2B 0"1' 1"BB Ter*enoid -/. Sa*onin -gm. E0tracti on Each 12 gm 0"1 0"2 0"2 0"2B 0"37 0"44 Sa*onin -/.

6oot /tem *e&ves

41"7B B6"13 4'"43

3'"' B3"BB 4B"34

C2"C3 CB"40 C3"61

3. &oncl!sion
3n the present stud+ le&-, stem &nd root sho!ed the presen#e o- bio&#tive #ompound su#h &s &l<&loids, -l&vonoids, terpenoids, s&ponins, et# " (his stud+ &lso le&ds to the -urther rese&r#h in the !&+ o- isol&tion &nd identi-i#&tion o- the &#tive #ompound -rom the le&-, stem &nd root o- Centella asiatica L. usin$ #hrom&to$r&phi# &nd spe#tros#opi# te#hni=ues"

Dry #atter -/.

)lant *art

,resh wt. -gm.

Dry wt. -gm.

Re4erences
@1A Phen$ J, J&n$ /Q" 4ntio8id&nt &#tivit+ &nd phenoli# #ompounds in sele#ted herbs" N 4$ri# >ood )hem 2001; 4C(11): B16B-B170" )&i QP, /un 2, )or<e D" 4ntio8id&nt &#tivit+ o- bet&l&ins -rom pl&nts o- the 4m&r&nth&#e&e" N 4$ri# >ood )hem 2003; B1('): 22''-22C4" /&l& 4, 6e#io 2,, Hiner 62, 2&neE /, (ournier D, /#hinell& H, 6ios N*" 4ntiin-l&mm&tor+ &nd &ntio8id&nt properties o- Deli#hr+sum it&li#um" N Ph&rm Ph&rm&#ol 2002; B4(3): 36B-371" 6i#e-7v&ns )4, 2iller NN, .ol!ell PH, .r&mle+ P2, Pridh&m N." (he rel&tive &#tivities o- Pl&nt-derived pol+phenoli# -l&vonoid" >ree r&di#&l 6es 1CCB; 22: 37B-3'3" 4sho<<um&r ,, 2&Eumder :I, Hupt& 2, /enthil<um&r HP, /elv&n ;(" 7v&lu&tion o- 4ntio8id&nt &nd >ree 6&di#&l /#&ven$in$ 4#tivities o- 08+stelm& es#ulentum in v&rious in vitro 2odels" N )omp 3nte$ 2ed 200'; B(1): 1-6"

@2A

Ac+nowledgement
4uthor ,r" /&n%&+ .ir&d&r, Prin#ip&l 3nvesti$&tor is $r&te-ul th&n<s to :H)1 Ne! ,elhi -or s&n#tioned the 2&%or 6ese&r#h Pro%e#t @>"No" 41-47C 2012(/6)A &nd &lso th&n<-ul to Prin#ip&l o- /hri )hh&tr&p&ti /hiv&%i )olle$e, 0mer$&, ,ist" 0sm&n&b&d, (2"/"), 3ndi& -or providin$ &ll ne#ess&r+ -&#ilities &nd en#our&$ement -or the present rese&r#h !or<"

@3A

@4A

@BA

4meri#&n Nourn&l o- *i-e /#ien#es 2013; 1(6): 243-247

247

@6A

;eer&pur ;P, Pr&bh&<&r I6, P&rih&r ;P, I&nd&di 26, 6&m&<rish&n& / et &l" >i#us r&#emos& /tem .&r< 78tr&#t: 4 Potent 4ntio8id&nt &nd & Prob&ble N&tur&l 6&dioprote#tor" 7vid .&sed )omplement 4ltern&t 2ed 200C; 6(3): 317-324" Iitts ,,, Qu&n Q;, Ji%e!i#<reme 4N, Du )" 4ntio8id&nt properties o- & North 4meri#&n $in$sen$ e8tr&#t" 2ol )ell .io#hem 2000; 20(3):1-10" 2uselR< N, H&r#R&-4lonso 2, 2&rtRn-*SpeE 2P, TelmiU<& 2, 6iv&s-HonE&lo N)" 2e&surement o- 4ntio8id&nt 4#tivit+ o- Jine )&te#hins, Pro#+&nidins, 4nto#+&nins &nd Pir&no&nto#+&nins" 3nt N 2ol /#i 2007; ': 7C7-'0C" J&n$ /Q, Ni&o D" )orrel&tion o- &ntio8id&nt #&p&#ities to o8+$en r&di#&l s#&ven$in$ enE+me &#tivities in bl&#<berr+" N 4$ri# >ood )hem 2000; 4': B672-B676"

@13A Phen$, )N" &nd ?in, *P" )hemi#&l #omponents o- )entell& &si&ti#& &nd their bio&#tives" Nourn&l o- )hinese 3nte$r&tive 2edi#ine 2007; B: 34'-3B1" @14A 4bdulr&hm&n >, 3n+&n$ /3, 4bb&h N, .ind& *, 4mos /, H&m&niel I (2004)" 7--e#t o- &=ueous le&- e8tr&#ts o3rvin$i& $&bonesis on $&strointestin&l tr&#ts o- rodents" 3ndi& N" 78p" .iol" 42:7'7-7C1" @1BA D&rborne N., Ph+to#hemi#&l methods: 4 Huide to 2odern te#hni=ues o- pl&nts 4n&l+sis" )h&pm&n &nd D&ll *ondon, :I" 1CC'" @16A D&rborne N." Ph+to#hemi#&l methods, *ondon" )h&pm&n &nd D&ll, *td" 1C73; 4C-1''" @17A .oh&m .4 &nd Io#ip&i-4b+&E&n 6" V>l&vonoids &nd #ondensed t&nnins -rom le&ves o- D&!&ii&n ;&##inium v&ti#ul&tum &nd ;" #&l+#iniumW P&#i-i# /#ien#e (1C74) 4': 4B'-463" @1'A 0bdoni .0 &nd 0#hu<o P0" VPh+to#hemi#&l studies &nd #omp&r&tive e--i#&#+ o- the #rude e8tr&#ts o- some Domost&ti# pl&nts in 7do &nd ,elt& /t&tes o- Ni$eri&W Hlob&l N" Pure 4ppl" /#i" (2001) ': 203-20'" @1CA >er$uson N2" 4 (e8t boo< o- Ph&rm&#o$nos+" 2&# 2il&n )omp&n+, Ne! ,elhi, 1CB6, 1C1"

@7A

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@10A /in$h"/", H&ut&m"4, /h&rm&"4 &nd .&tr&"4 )entell& &si&ti#& (*"): 4 pl&nt !ith immense medi#in&l potenti&l but thre&tened, 3ntern&tion&l %ourn&l o- ph&rm&#euti#&l s#ien#es revie! &nd rese&r#h 2010; 4(2): C-17" @11A *oise&u, 4" &nd 2er#ier, 2" )entell& &si&ti#& &nd s<in #&re" )osmeti#s &nd (oiletries 2&$&Eine 2000; 11B: 63- 67" @12A .rin<h&us .", *indner 2", /#hupp&n ," &nd D&hn 7"H", )hemi#&l, ph&rm&#olo$i#&l &nd #lini#&l pro-ile o- the 7&st 4si&n medi#&l pl&nt )entell& &si&ti#&, Ph+tomedi#ine, 7(B), 427-4' (2000)