Phân Lập Các Thành Phần Có Khả Năng Chống Oxy Hóa Trong Lá Chùm Ngây

Y Hc TP. H Ch Minh * Tp 16 * Ph bn ca S 1 * 2012 Nghin cu Y hc

Chuyn Y Hc C Truyn 163

PHN LP CC THNH PHN C TC DNG CHNG OXI HA

TRONG L CHM NGY (MORINGA OLEIFERA LAM.)

Salihah*, Bi Th Vinh*, Trn Cng Lun*

TM TT

Mc tiu nghin cu: Xc nh v phn lp cc thnh phn ha hc c tc dng chng oxi ha trong

l Chm ngy

i tng Phng php nghin cu: L Chm ngy ti thu hi ti Tri tn, An Giang c chit

bng phng php ngm vi cn 96% thu c cao cn ton phn. Chit phn on vi dung mi c

phn cc tng dn: Dietyl eter, cloroform, etyl acetat, thu c cc cao phn on. nh lng phenolic

ton phn trong cao cn ton phn v cc cao phn on bng phng php Folin Ciocalteu vi cht

chun l acid gallic. Xc nh cc thnh phn c hot tnh oxi ha trong cao etyl acetat bng phng php

sc k lp mng v pht hin bng cch phun thuc th DPPH. Sau phn lp cc thnh phn c hot

tnh chng oxi ha bng phng php sc k nhanh ct kh v sc k ct c in. Xc nh cu trc ca

cc cht phn lp c bng cc k thut ph NMR mt chiu v hai chiu, ph MS v so snh vi cc

thng s ph NMR ca cc hp cht cng b.

Kt qu: T 10 kg l ti, thu c 600g cao cn ton phn. Chit phn on thu c 168 g cao eter,

2,63 g cao cloroform, 28,4 g cao etyl acetat v 120g cao nc. Hm lng phenolic ton phn trong cao cn

ton phn l 108,011 g/mg. Hm lng phenolic ton phn trong cao eter, cao cloroform, cao etyl acetat v

cao nc ln lt l 44,528 g/mg, 110,914 g/mg, 271,822 g/mg v 90,881 g/mg tnh theo acid gallic

chun. Trong phn on etyl acetat, xc nh c 6 vt cht c hot tnh chng oxi ha vi gi tr Rf ln

lt l 0,45; 0,29; 0,27; 0,16; 0,12; 0,1 vi h dung mi: Etyl acetat metanol nc (100: 17: 13). T

phn on etyl acetat phn lp c 2 hp cht c hot tnh chng oxi ha l ch yu l isoquercitrin (200

mg) v vitexin (25 mg).

Kt lun: L Chm ngy cha nhiu hp cht phenolic c hot tnh chng oxi ha. Trong phn

lp c 2 hp cht c hot tnh chng oxi ha ch yu l vitexin v isoquercitrin. Vitexin l mt flavon C-

glycosid c tm thy ln u tin t l cy ny.

T kho: chm ngy, chng oxy ho.

ABSTRACT

ISOLATING THE ANTIOXIDANT COMPOUNDS FROM LEAVES OF MORINGA OLEIFERA

LAM.

Salihah, Bui The Vinh, Tran Cong Luan

* Y Hoc TP. Ho Chi Minh * Vol. 16 - Supplement of No 1 2012: 163 - 168

Objects: To define and isolate the antioxidants from leaves of Moringa oleifera Lam.

Materials and Methods: Moringa oleifera fresh leaves collected from Tri ton - An Giang province were

extracted with 96% alcohol. The crude alcohol extract was separated using three solvents: Diethyl ether,

chloroform, and ethyl acetate. Total phenolic contents of extracts were measured with Folin-Ciocalteu reagent

using gallic acid as a standard.

Defining the antioxidants from ethyl acetate extract using thin layer chromatography with DPPH reagent.

Trung tm Sm v Dc liu Tp. HCM Vin Dc Liu

Tc gi lin lc: PGS.TS. Trn Cng Lun, T: 0903671323, Email: [email protected]

Nghin cu Y hc Y Hc TP. H Ch Minh * Tp 16 * Ph bn ca S 1 * 2012


Then, those antioxidants were isolated by dry-column flash chromatography method and open- column

chromatography method. The structure elucidations were based on analyses of spectroscopic data including 1D-

and 2D-NMR, and MS.

Results: 600 g crude alcohol extract macerated from 10 kg fresh leaves of M. oleifera was fractionated to 168

g diethyl ether, 2.63 g chloroform, 28.4 g ethyl acetate and 120 g water fraction, respectively. Total phenolic

contents of extracts were (1)- alcohol crude extract: 108.011 g GAE/mg, (2)- diethyl ether extract: 44.528 g

GAE/mg, (3)- chloroform extract: 110.914 g GAE/mg, (4)- ethyl acetate extract: 271.822 g GAE/mg, (5)-

water extract: 90.881 g GAE/mg. Six spots on chromatogram having antioxidant activity were defined in ethyl

acetate fraction with Rf values of 0.45, 0.29, 0.27, 0.16, 0.12 and 0.1 with solvent system as etyl acetat metanol

water (100: 17: 13). Two antioxidants isolated from ethyl acetate fraction were isoquercitrin (200 mg) and

vitexin (25 mg).

Conclusion: Two antioxidants of phenolic compounds islolated from M. oleifera leaves are vitexin and

isoquercitrin, in which vitexin is found from those leaves at the first time.

Keywords: Moringa oleifera, antioxidant.

T VN

Chm ngy (Moringa oleifera Lam.) c gi

l cy Thn Diu v n cha rt nhiu cht

dinh dng quan trng nh: Vitamin C, -

caroten, protein, cc acid amin, v nhiu hp

cht c tc dng sinh hc nh: Zeatin, flavonoid

(quercetin, kaempferol...), -sitosterol, axit

caffeoylquinic, niazirinin, niaziminin A v B,

benzyl isothiocyanat...(7,8). Chnh v th, n

c nghin cu v ng dng rt nhiu trong

thc phm, y dc hc ti nhiu quc gia trn

th gii nh: n , Pakistan, Ghana, Malaysia,

Thi Lan(4,6). Nhng ti Vit Nam, Chm ngy

cha c quan tm v nghin cu nhiu,

chng ch mc hoang ngoi t nhin, sng ri

rc mt s vng t Qung Nam n Ph

Quc, cc b phn ca cy nh qu, l non, hoa

cc nhnh non ch dng lm rau n trong dn

gian(8). Gn y ngi ta cng quan tm v t

chc trng mt s ni nh Tri tn - An Giang,

Long Khnh - ng Nai v c mt s ch

phm dng thc phm chc nng c lu

hnh trn tr trng. Nhm gp phn pht trin

cy Chm ngy nh l mt ngun nguyn liu

tim nng cho cc dng thuc hay thc phm

chc nng trong chm sc sc khe cho cng

ng, chng ti tin hnh phn lp cc hp

chnh c tc dng chng oxy ha trong l Chm

ngy.

VT LIU V PHNG PHP

L Chm ngy c thu hi ti Tri Tn, An

Giang vo u thng 8/2010. Mu s dng bao

gm c cung l v l cht cn ti.

Chit xut dc liu

10 kg l ti c chit bng phng php

ngm nhit phng, vi dung mi chit l

cn 96%, theo t l 6: 1 v chit 6 ln. C di p

sut gim thu c cao cn ton phn, chit

phn on ln lt vi cc dung mi c

phn cc tng dn: Dietyl eter, cloroform, etyl

acetat, nc thu c cc cao phn on: Cao

eter, cao cloroform, cao etyl acetat v cao nc.

nh lng phenolic ton phn

Bng phng php Folin Ciocalteu theo

Waterman v Mole (1994)(2). Dng ng chun

acid gallic vi giai mu 10 g, 20 g, 30 g, 40

g, 50g, da v phng trnh ng chun

xc nh hm lng phenolic ton phn trong

mu th. Hm lng phenolic ton phn c

tnh bng g ng lng acid gallic c trong 1

mg mu th (g GAE/ mg). Tin hnh th

nghim nh sau: Ht mt th tch xc nh mu

(Cht chun acid gallic hoc mu cn nh

lng c pha long nng thch hp) cho

vo bnh nh mc 10 ml.Thm 6 ml nc ct,

lc u. Cho vo bnh 0,5 ml thuc th Folin-

Ciocalteu, lc u. yn trong 5 pht.Thm 1,5



ml Na2CO3, lc u. Thm nc cho va

10ml. yn trong ti 2h. Sau tin hnh o

hp thu bc sng 758 nm.

Xc nh cc thnh phn c hot tnh

chng oxi ha trong phn on etyl acetat

Bng phng php sc k lp mng vi h

dung mi khai trin: etyl acetat metanol

nc (100: 17: 13)(1). Sau khi khai trin, bn mng

c quan st di n UV 254 nm v UV 365

nm. Sau phun thuc th DPPH 0,05% ln b

mt ca bn mng v nhit phng trong

10 pht. Nhng thnh phn c hot tnh chng

oxi ha s to nhng vch mu vng trn nn

tm ca bn mng, do cht chng oxi ha lm

mt mu tm ca thuc th DPPH. Chng

dng l vitamin C. So snh bng mt thng

mt mu ca vch chng dng v cc vch

c hot tnh khc xc nh mc mnh yu

ca hot tnh. Nhng vch c hot tnh chng

oxi ha c ghi nhn li bng gi tr Rf ca

chng.

Sc k nhanh ct kh (7)

S dng phu Buchner thnh cao ng

knh trong 10 cm, bnh tam gic, my ht chn

khng p sut 70 mmHg. Cht hp ph l

silicagel ht va 40 60 m. Chiu cao ct cht

hp ph 5 cm. Np mu dng bt kh, khi

lng mu 20 g. H dung mi ra gii:

Cloroform 100%, etyl acetat 100%, etyl acetat

metanol (50: 50). Th tch phn on ra gii:

500 ml. Kim tra cc phn on bng sc k lp

mng vi h dung mi etyl acetat metanol

nc (100: 17: 13). Cc phn on ging nhau

c gp chung. C n cn v cn khi lng.

Sc k ct c in

Cht hp ph l silicagel ht va (40 60

m). Khi lng cht hp ph 350g. Quy cch

ct 4 x 50 cm. Khi lng mu 6 g. Np mu

bng phng php nhi bt kh. Dung mi ra

gii l etyl acetat: metanol. Tc dng 20 git/

pht. Th tch mi phn on 20 ml.

Xc nh cu trc ca cc cht phn lp

c bng ph 1H-NMR, 13C-NMR, DEPT,

COSY, HSQC, HMBC, v ph MS.

KT QU V BN LUN

Chit xut dc liu

T 10 kg l Chm ngy ti, chit bng cn

96% thu c 600 g cao cn ton phn, m

cao l 19,8 %. Hiu sut chit l 5,88 %.

Kt qu lc phn on thu c khi lng

cao theo bng 1

Bng 1: Kt qu thu cao phn on

Phn on Khi lng cao (g)

m cao (%)

Hiu sut chit

Cao eter 168 10,53 28%

Cao cloroform 2,63 15,62 0,44 %

Cao etyl acetat 28,40 18,25 4,7 %

Cao nc 120 19,42 20 %

nh lng phenolic ton phn

Hnh 1: Phng trnh ng chun acid gallic

Da vo ng chun acid gallic xc nh

hm lng phenolic ton phn vi phng

trnh hi quy: y = 0,014x 0,0021 v R2 = 0,9955

Bng 2: Hm lng phenolic ton phn trong cao

cn ton phn v cao phn on

Mu Khi

lng cao (mg)

OD 758 nm Hm lng

phenolic ton phn (g GAE/mg)

Cao ton phn 0,4 0,483 108,011

Cao eter 0,4 0,221 44,528

Cao cloroform 0,4 0,522 110,914

Cao etyl acetat 0,1 0,309 271,822

Cao nc 0,4 0,408 90,881

Ghi ch: GAE: Gallic acid equivalent (tng ng vi

acid gallic)



Hm lng phenolic ton phn trong cao

cn ton phn l 108,011 g GAE/ mg. v trong

cc cao phn on: Cao eter, cao cloroform, cao

etyl acetat v cao nc ln lt l 44,528 g

GAE/mg, 110,914 g GAE/mg, 271,822 g

GAE/mg v 90,881 g GAE/mg. Trong cao

etyl acetat l phn on c hot tnh chng oxi

ha nht v c la chn tip tc phn lp

cc cht c hot tnh chng oxi ha trong l

Chm ngy.

Xc nh cc thnh phn c hot tnh chng oxi ha trong phn on etyl acetat

UV 254 nm UV 365 nm Thuc th FeCl3 Thuc th DPPH 0,05%

Hnh 2: Kt qu sc k lp mng phn on etyl acetat, h EtOAc MeOH H2O (100:17:13)

Khi bt u phun thuc th DPPH ln bn

mng, vitamin C ngay lp tc lm mt mu ca

DPPH v to vt vng trn sc . i vi cc

vt cht trong phn on etyl acetat cng c tnh

trng tng t, khng cn 10 pht m cc vt

ngay lp tc lm mt mu DPPH. iu ny cho

thy hot tnh chng oxy ha ca cc thnh

phn trong phn on ny l rt cao. Trong

phn on etyl acetat c 6 vt cht c mu vng,

th hin hot tnh chng oxy ha. Chng c gi

tr Rf ln lt l 0,45 ; 0,29 ; 0,27 ; 0,16 ; 0,12; 0,1

h EtOAc MeOH H2O (100:17:13) v u hin

mu vi thuc th FeCl3.

Sc k nhanh ct kh

Bng 3: Kt qu cc phn on thu c t sc k nhanh ct kh

H dung mi Phn on tp hp Phn on Khi lng cao

(g) Thnh phn Ghi ch

CHCl3

C1 1 6 0,2 Tp

C2 7 10 0,3044 Tp C cc hp cht phenolic

km phn cc

EtOAc

E1 11 1,0 Vt X1 + tp

E2 12 1,2552 X2

E3 13 0,5537 X2 + tp

E4 14 30 8,5278 X3 + X4 Thnh phn c hot tnh

chng oxy ha

EtOAc MeOH (5: 5)

EM 31 36 8,3309 Thnh phn c hot tnh

chng oxy ha

Cao EtOAc Cao EtOAc Cao EtOAc Cao EtOAc Vitamin C



Phn on E4 c chn tin hnh sc k

ct c in, phn tch cc hp cht c hot tnh

chng oxi ha trong l Chm Ngy.

Sc k ct c in

Bng 4: Cc phn on thu c t sc k ct c in

H dung mi

Phn on tp hp

Phn on

Khi lng cao (g)

Thnh phn

EtOAc 100%

MO1 1 40 0,56 Tp

MO2 41 49 0,35 Tp + cht

1

MO3 50 54 0,11 cht I + t

tp

MO4 55 80 0,65 cht I + cht II

MO5 81 160 2,19 cht II + tp pha

di

EtOAc MeOH (95:5)

MO6 136 208 1,55 cht II

MO7 209 258 1,92 cht II + cht III

T phn on MO3, qua m, xut hin

ta mu vng lng di y. Tch ring ta v

ra ta bng metanol, thu c cht I. T phn

on MO5 v MO6 qua m, xut hin kt

tinh hnh kim mu vng. Tch ring kt tinh,

lm sch bng cch cho ti kt tinh trong dung

mi etyl acetat, thu c cht II

Xc nh cu trc ca hp cht phn lp

c

Cht I v cht II c o v gii ph NMR

(ph H, ph C, DEPT, HSQC, COSY, HMBC) v

khi ph MS. Da vo kt qu ph NMR mt

chiu v 2 chiu, d on cu trc ca cht I l

vitexin v cht II l isoquercitrin. Ph H v ph

C ca cht I v cht II c i chiu vi ph

ca cht chun c cng b.

Bng 5: Thng s ph 1H (500 MHz, DMSO-d6) v 13C-NMR (125 MHz, DMSO-d6) ca cht I i chiu

vi vitexin(9)

Cht I Vitexin

C 13C 1H 13C 1H

(ppm) (ppm) (ppm) (ppm)

2

163,85

102,37

181,97

155,90

98,08

164,98

Cht I Vitexin

162,49

104,55

160,32

103,96

121,54

128,81

115,73

161,03

115,73

128,81

73,31

70,82

78,62

70,54

81,72

61,26

3 102,37 6,77 102,51 6,94 (1H, s, H-3)

4 181,97 182,73

5 155,90 13,16 155,64 13,17 (1H, s, 5-OH)

6 98,08 98,45 6,44 (1H, s, H-6)

7 162,49 162,31 10,83(1H, s, 7-OH)

8 104,55 104,56

9 160,32 160,28

10 103,96 104,07

1

1

121,54 122,07

2 128,81 8,03 128,99 8,26 (1H, d, J = 8,7

Hz, H-2)

3 115,73 6,90 115,01 7,05 (1H, d, J= 8,7

Hz, H-3)

4 161,03 161,32 10,35 (1H, s, 4-OH)

5 115,73 6,88 115,01 7,05 (1H, d, J= 8,7

Hz, H-5)

6 128.81 8,01 128,99 8,26 (1H, d, J = 8,7

Hz, H-6)

1 73,31 4,93 73,93 4,94 (1H,d, J = 9,8

Hz, H-1)

2 70,82 71,03

3 78,62 79,01

4 70,54 70,20

5 81,71 81,29

6 61,26 61,36

Bng 6: Kt qu ph 1H (500 MHz, DMSO-d6) v 13C-

NMR (125 MHz, DMSO-d6) ca cht II i chiu vi

isoquercitrin (3) Cht II isoquercitrin

C 13C 1H 13C 1H (ppm) (ppm) (ppm) (ppm) 2 156,16 156,0 3 133,35 133,2



Cht II isoquercitrin 4 177,39 177,2 5 161,18 161,0

6 98,58 6,20 98,6 6,18 (1H, d, J = 1,8

Hz, H-6) 7 164,03 164,0

8 93,42 6,40 93,5 6,39 (1H, d, J = 1,8

Hz, H-8) 9 156,27 156,1 10 103,95 103,9 1 1

121,15 121,0

2 115,15 7,56 115,1 7,56 (1H, br s, H-2) 3 144,72 144,6 4 148,37 148,3

5 116,19 6,83 116,1 6,83 (1H, d, J = 9,0

Hz, H-5)

Cht II isoquercitrin

6 121,51 7,58 121,5 7,58 (1H, d, J = 9,0

Hz, H-6)

1 100,96 5,45 100,8 5,45 (1H, d, J = 6,9

Hz, H-1) 2 74,06 74,1 3 76,49 76,5 4 69,94 70,0 5 77,42 77,5 6 60,97 61,0

Cu trc cht I l vitexin (C21H20O10: (1S)-1,5-anhydro-

1-[5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-

chromen-8-yl]-D-glucitol) v cht II l isoquercitrin

(C21H20O12: 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-

oxo-4H-chromen-3-yl -D-glucopyranosid).

Cu trc ca cht I - vitexin Cu trc ca cht II - isoquercitrin

Hnh 3: Cu trc ca vitexin v isoquercitrin

KT LUN

L Chm ngy cha nhiu hp cht c

hot tnh chng oxi ha, cc hp cht ny lin

quan n nhm hp cht phenolic. Trong

hai hp cht flavonoid c hot tnh chng oxi

ha ch yu ca l c xc nh l vitexin

v isoquercitrin. Vitexin l mt flavon C-

glycosid c phn lp t l cy ny ln u

tin.

TI LIU THAM KHO 1. Bhattarai H. D., Babita P., Hong S.G., Lee H.K., Yim J.H.

(2008), Thin layer chromatography analysis of antioxidant

constituents of lichens from Antarctica, Journal of Natural

Medicine, 62, pp. 481 484.

2. Chumark P., Panya K., Yupin S., Srichan P., Noppawan M.

(2008), The in vitro and ex vitro antioxidant properties,

hypolipidaemic and antitherosclerotic activities of water

extract of Moringa oleifera Lam. leave, Journal of

Ethnopharmacology, 119, pp. 439 436.

3. Deepralard K., Kawanishi K., Moriyasu M., Thitima P.,

Suttisri R. (2009), Flavonoid glycosides from the leave of

Uvaria rufa with advanced glycation end products

inhibitory activity, Thai J. Pharm. Sci., 33, pp. 84 90.

4. Fahey J.W. (2005), Moringa oleifera: A Review of the Medical

Evidence for Its Nutritional, Therapeutic, and Prophylactic

Properties. Trees for Life Journal,1: pp. 5.

5. Monzon R.B. (1995), Traditional medicine in the treatment of

parasitic diseases in the Philippines, Southeast Asian Journal

of Tropical Medicine and Public Health, 26(3): pp. 421-428.

6. Sabale V., Patel V., Paranjape A., Arya C., Sakarkar S. N. and

Sabale P.M. (2008), Moringa Oleifera (Drumstick): An

Overview, Pharmacognosy Reviews, 2(4), pp. 7-13.

7. Trn Hng (2006), Phng php chit xut dc liu, B mn

Dc liu, Khoa Dc- i hc Y Dc, Tp. HCM, tr. 119 -

127.

8. V Vn Chi, (1999), T in cy thuc Vit Nam, NXB Y Hc,

tr. 248.

9. Zhou X., Peng J., Fan G.(2005), Isolation and purification of

flavonoid glycosides from Trollius ledebouri using high-speed

counter-current chromatography by stepwise increasing the

flow-rate of the mobile phase, J Chromatoqr A., 1092 (2), pp.

216 221.

Documents

Phân Lập Các Thành Phần Có Khả Năng Chống Oxy Hóa Trong Lá Chùm Ngây