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PHN LP V NHN DANH CU TRC HAI FLAVONOL T DCH CHIT ETHYL ACETATE CA CY C LO-EUPATORIUM ODORATUM L.
Ng Quc Lun1, Nguyn Ngc Chu2 v Nguyn Ngc Hnh3

ABSTRACT
From the ethyl acetate extracts of the aerial parts of Eupatorium odoratum L. from Phu Yen province, two of flavonols (rhamnocitrin and rhamnetin) were isolated. Their structures were interpreted from spectra including IR, 1H-NMR, 13C-NMR, HSQC, HMBC, MS and based on published data. These flavonols were tested for antioxidant activity by DPPH method. The result showed that their antioxidant activity is similarly strong. Keywords: Eupatorium odoratum L., Chromolaena odorata L. Title: Isolation and identification of two flavonols from ethyl acetate extracts of Eupatorium odoratum L.

TM TT
T dch chit ethyl acetate phn trn mt t ca cy C Lo Ph Yn, chng ti c lp c hai flavonol l rhamnocitrin v rhamnetin. Cu trc cc cht ny c xc nh bng cc phng php ph hin i nh IR, 1H-NMR, 13C-NMR, HSQC, HMBC, MS v so snh vi ti liu cng b. Cc flavonol trn c th hot tnh khng oxi ha bng phng php DPPH. Kt qu cho thy hai cht ny c tnh khng oxi ha mnh tng ng nhau. T kha: Cy C Lo, Cy Cng Sn

1 GII THIU Trong hai bi bo Mt s kt qu nghin cu thnh phn ha hc ca tinh du v flavonoid trong cy C Lo (Ng Quc Lun, Lm Thanh Phong, Nguyn Ngc Hnh, Tp ch khoa hc, Trng i hc Cn Th, S 6, 2006) v Phn lp v nhn danh cu trc mt chalcone t dch chit ethyl acetat ca cy C Lo (Ng Quc Lun, Nguyn Ngc Chu, Nguyn Ngc Hnh, Tp ch khoa hc, Trng i hc Cn Th, S 08, 2007), chng ti bo co mt s kt qu nghin cu bc u v thnh phn ha hc ca tinh du v flavonoid t cy C Lo mc Ph Yn. Trong bi bo ny, chng ti tip tc trnh by mt s kt qu kho st thnh phn ha hc v th hot tnh sinh hc ca hai flavonol c lp t dch chit ethyl acetate. 1.1 Nguyn liu Ton b phn trn mt t ca cy C Lo c thu hi khi cy bt u ra hoa vo thng 12-2005 ti cc vng ven i ni thuc x Ha Hip Nam, huyn ng Ha, tnh Ph Yn.
1 2

Khoa S Phm , Trng i hc Cn Th Khoa Khoa hc c bn, Trng Cao ng Cng ng ng Thp 3 Vin Cng ngh Ha hc, Vin Khoa hc v cng ngh Vit Nam

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1.2 Phng php chit xut, c lp Nguyn liu ti (100 kg) sau khi tch tinh du bng phng php li cun hi nc c tip tc trch nng vi 320 lt EtOH 50 trong 3 gi, sau c c n cn 8 kg dch chit st. Dch ny c lc chit vi ethyl acetate (EtOAc) v c loi dung mi di p sut km thu c cao EtOAc (139 g). T cao EtOAc (129 g), tin hnh sc k nhanh trn ct silica gel vi h dung mi gii ly (XDM*, EtOAc) c phn cc tng dn. Kt qu thu c 15 phn on (LA1, LA2, v LA15). Phn on LA2 (8 g) c tip tc tin hnh sc k ct silica gel ln 2 cng vi h dung mi gii ly c phn cc tng dn l (XDM, EtOAc). Kt qu thu c 30 phn on (LA2-1, LA2-2, v LA2-30) Ti phn on LA2-2 (dung mi gii ly XDM: EtOAc = 9:1) thu c cn mu vng c vt chnh vi Rf = 0,39 (PE:EtOAc = 1:1). Kt tinh li trong EtOAc thu c cht rn dng bt mu vng rm (hnh 1, 2), k hiu l LA2-2 (20 mg).

Hnh 1

Hnh 2

Phn on LA6 (9 g) c tip tc tin hnh sc k ct silica gel ln 2 cng vi h dung mi gii ly c phn cc tng dn l (XDM, EtOAc). Kt qu thu c 30 phn on (LA6-1, LA6-2, v LA6-30) Ti phn on LA6-9 (dung mi gii ly XDM: EtOAc = 7:3) thu c cn mu vng c vt chnh vi Rf = 0,21 (PE:EtOAc = 1:1). Kt tinh li trong MeOH thu c cht rn dng bt mu vng ngh (hnh 3, 4), k hiu l LA6-9 (25 mg).
* Xng dung mi hay dung mi cao su, mt loi petroleum ether c khong si 79-96C

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Hnh 3 1.3 Phng php nhn dng cu trc

Hnh 4

im tan chy c o trn my Electrothemal 9100 (U.K), mao qun khng hiu chnh. Ph hng ngoi c o trn my VECTOR 22, dng vin nn KBr. Ph UV-VIS c do trn my UV-2450 (Japan). Cc ph 1H-NMR, 13C-NMR, HMQC, HMBC c ghi trn my Bruker Avance 500 MHz, dch chuyn ha hc () c tnh theo ppm, hng s tng tc (J) tnh bng Hz. Ph khi lng c o trn my 1100 series LC/MS Trap Agilent, sc k lp mng s dng bn nhm silica gel 60F254 (Merck) trng sn dy 0,2 mm. 1.4 Phng php th hot tnh khng oxi ha Trong nghin cu ny chng ti chn phng php gc t do bn DPPH (2,2diphenyl-1-picrylhydrazyl) nh sau: - Ha tan cht tinh khit trong DMSO nng : 500 g/ml. - DPPH ha tan trong dung mi ethanol nng 250M. - Cho 8 ml dung dch DPPH nng 250M phn ng vi 2 ml dung dch cht tinh khit nng 500 g/ml. - Dung dch c lc u, thc hin phn ng iu kin nhit phng trong thi gian 30 pht, ri em mu o hp thu bc sng = 517 nm. - Dy chun v mu trng c thc hin trong iu kin tng t nh trn. 2 KT QU NGHIN CU V THO LUN 2.1 Nhn danh cu trc flavonoid 2.1.1 LA2-2 LA2-2 kt tinh trong EtOAc thu c cht rn dng bt, mu vng rm, c nhit nng chy mp = 217-218C (EtOAc), sc k bn mng trong h dung mi (PE:
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EtOAc = 1:1), hin vt bng thuc th H2SO4 10% trong EtOH cho vt mu da cam c Rf = 0,39. - Ph 1H-NMR, (DMSO, ppm) cho 8 mi tn hiu proton: + 1 mi n ti 3,86: 3 proton ca 1 nhm OCH3 gn vo vng benzene. + 4 mi trong khong 6,34-8,09 l tn hiu ca cc proton ca nhm CH k ni i, cho thy c 2 vng benzene, trong : * Vng A: 2 proton ghp cp meta ti 6,35 (1H, d, J= 2,0 Hz) v 6,74 (1H, d, J= 2,0 Hz). * Vng B: 2 mi i l 4 proton cho bit c 2 cp proton v tr i xng nhau trn vng benzene nn 2 cp proton tng ng di ha hc, chng ghp cp orto ti 8,08 (1H, d, J= 8,5 Hz) v 6,92 (1H, d, J= 9,0 Hz). + 1 mi n ti 12,47: 1 proton ca nhm OH. + 2 mi n cn li ti 9,49 v 10,11 c th l 2 proton ca 2 nhm OH. Vy LA2-2 c tt c 12 proton. - Cc ph 13C-NMR (DMSO, ppm) cho thy cht LA2-2 c 14 mi tn hiu carbon. + 8 mi trong khong 104,01-164,88: 8 carbon t cp (104,01; 121,56; 135,94; 147,23; 156,07; 157,27; 160,35; 164,88). + 1 mi ti 55,99: 1 carbon ca nhm OCH3 gn vo vng benzene. + 1 mi ti 176,00: 1 carbon ca nhm C=O c trng ca hp cht flavone. + 4 mi trong khong 91,99-129,55: 6 carbon loi CH k ni i ( 91,99; 97,43; 115,43; 129,55). Vy LA2-2 c tt c 16 nguyn t carbon (trong c 2 cp carbon tng ng di ha hc). Cn c vo cc c tnh ph NMR ca LA2-2 c th d on cht ny c khung flavone. Ngoi 5 nguyn t oxy xut hin theo cc nhm trn ph NMR, LA2-2 cn c thm 1 nguyn t oxy. Vy LA2-2 c 6 nguyn t oxy. Nh vy, LA2-2 c cng thc phn t l C16H12O6, phn t khi l 300 vC; bt bo ha l 11, trong : vng pyron chim 4 bt bo ha, vy cn 7 bt bo ha c th l ca 2 vng benzene (1 vng ghp 1 cnh vi vng pyron v 1 vng gn vi vng pyron qua dy ni), ph hp vi c cu khung flavone (Hnh 5). Qui np cc di ha hc vo cc v tr trn khung flavone (Bng 1) v kim tra li bng cc ph hai chiu HMBC (DMSO, ppm) cho thy s tng tc gia H vi C ti cc v tr C m H c th tng tc: H6C6, C8, C10, C5, C7; H8C8, C6, C10, C9, C7, H2C2, C6, C2, C4; H3C3, C5, C1, C4 ; H5C5, C1, C3, C4; H6C6, C2, C2, C4 ; 7OCH3C7; 5-OHC6, C10, C5.
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Bng 1: D liu ph 1H-NMR, 13C-NMR, v HMBC ca LA2-2

V tr C/H 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7OCH3 5-OH

H-NMR ( ppm, J = Hz)

13

C-NMR ( ppm) 147,23 135,94 176,00 160,35 97,43 164,88 91,99 156,07 104,01 121,56 129,55 115,42 159,27 115,42 129,55 55,6

Loi carbon =C< =C< >C=O =C< =CH=C< =CH=C< =C< =C< =CH=CH=C< =CH=CH-CH3

HMBC H13C

6,35 (1H; d; J = 2,0) 6,74 (1H; d; J = 2,0)

H6C6, C8, C10, C5, C7 H8C8, C6, C10, C9, C7

8,08 (2H, d, J= 8,5 Hz) 6,92 (2H, d, J= 9,0 Hz) 6,92 (2H, d, J= 9,0 Hz) 8,08 (2H, d, J= 8,5 Hz)
3,86 (3H, s) 12,47 (1H, s)

H2C2, C6, C2, C4 H3C3, C5, C1, C4 H5C5, C1, C3, C4 H6C6, C2, C2, C4 7OCH3C7 5-OHC6, C10, C5

Tm li, da vo cc kt qu ph cng hng t ht nhn, cc c trng vt l v so snh vi cc ti liu cng b (David S. Seigler and E. Wollenweber, 1983; Maria Rose Jane R. Albuquerque et al., 2006), chng ti nhn danh cht LA2-2 l rhamnocitrin (7-O-methylkaempferol hay 3,5,4- trihydroxy-7-methoxyflavone) c cng thc cu to nh hnh 5.
H 3CO
6 10 5 8 7 9

O
4

6' 2 1'

5' 4'

OH

2'

3'

OH

OH

Rhamnocitrin: C16H12O6
Hnh 5: Cng thc cu to ca LA2-2

2.1.2 LA6-9 LA6-9 kt tinh trong MeOH thu c cht rn dng bt, mu vng ngh, c nhit nng chy mp = 263-264C (MeOH), sc k bn mng trong h dung mi (PE: EtOAc = 1:1), hin vt bng thuc th H2SO4 10% trong EtOH cho vt mu da cam c Rf = 0,21. - Ph 1H-NMR, (DMSO, ppm) cho 8 mi tn hiu proton:
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+ 1 mi n ti 3,86: 3 proton ca 1 nhm OCH3 gn vo vng benzene. + 5 mi trong khong 6,34-7,72 l tn hiu ca cc proton ca nhm CH k ni i, trong : * 3 mi n: 3 proton (6,33; 6,68; 7,71). * 2 mi i: 2 proton ghp cp orto ti 6,88 (1H, d, J= 8,5 Hz) v 7,57 (1H, d, J= 8,0 Hz). + 1 mi n ti 12,47: 1 proton ca nhm OH. + 1 mi rng ti 9,43 c th l 3 proton ca 3 nhm OH. Vy LA6-9 c tt c 12 proton. - Ph 13C-NMR (DMSO, ppm) cho thy cht LA6-9 c 16 mi tn hiu carbon. + 9 mi trong khong 103,99-164,89: 9 carbon t cp (103,99; 121,87; 136,01; 145,07; 147,29; 147,83; 156,06; 160,37; 164,89). + 1 mi ti 56,00: 1 carbon ca nhm OCH3 gn vo vng benzene. + 1 mi ti 175,95: 1 carbon ca nhm C=O c trng ca hp cht flavone. + 5 mi trong khong 91,88-120,04: 5 carbon loi CH k ni i (91,88; 97,42; 115,24; 115,57; 120,04). Vy LA6-9 c tt c 16 nguyn t carbon Cn c vo cc c tnh ph NMR ca LA6-9 c th d on cht ny c khung flavone. Ngoi 6 nguyn t oxy xut hin theo cc nhm trn ph NMR, LA6-9 cn c thm 1 nguyn t oxy. Vy LA6-9 c 7 nguyn t oxy. Nh vy, LA6-9 c cng thc phn t l C16H12O7, phn t khi l 316 vC; bt bo ha l 11, trong vng pyron chim 4 bt bo ha, vy cn 7 bt ha c th l ca 2 vng benzene (1 vng ghp 1 cnh vi vng pyron v 1 vng gn vi vng pyron qua dy ni), ph hp vi c cu khung flavone (Hnh 6). Qui np cc di ha hc vo cc v tr trn khung flavone (Bng 2) v kim li bng cc ph hai chiu HSQC v HMBC: - Ph hai chiu HSQC (DMSO, ppm) cho thy s tng tc gia H vi C ti v tr C m H gn vo: H6C6; H8C8; H2C2; H5C5; H6C6. - Ph hai chiu HMBC (DMSO, ppm) cho thy s tng tc gia H vi C ti cc v tr C m H c th tng tc: H6C6, C8, C10, C5, C7; H8C8, C6, C10, C2, C7; H2C2, C6, C3, C4; H5C5, C1, C3, C4; H6C6, C2, C4 ; 7OCH3C7; 5OHC6, C10, C5.

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Bng 2: D liu ph 1H-NMR, 13C-NMR, HSQC v HMBC ca LA6-9

V tr C/H 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7OCH3 5-OH

H-NMR ( ppm ,J = Hz)

13

C-NMR ( ppm) 156,06 136,01 175,95 160,37 97,42 164,89 91,88 147,29 103,99 121,87 115,24 145,07 147,83 115,57 120,04 56,00

Loi carbon =C< =C< >C=O =C< =CH=C< =CH=C< =C< =C< =CH=C< =C< =CH=CH-CH3

HMBC H13C

6,33 (1H; s) 6,68 (1H;s)

H6C6, C8, C10, C5, C7 H8C8, C6, C10, C2, C7

7,71 (1H, s)

H2C2, C6, C3, C4

6,88 (1H, d, J= 8,5) 7,57 (1H, d, J= 8,0)

3,86 (3H, s) 12,47 (1H, s)

H5C5, C1, C3, C4 H6C6, C2, C4 7OCH3C7 5-OHC6, C10, C5

Tm li, da vo cc kt qu ph cng hng t ht nhn, cc c trng vt l v so snh vi ti liu cng b (Toan Thang Phan, Lingzhi Wang, Patrick See, Renee Jacqueline Grayer, Sui Yung Chan and Seng Teik Lee, 2001), chng ti nhn danh cht LA6-9 l rhamnetin (7-methoxyquercetin hoc 3,5,3,4tetrahydroxy-7-methoxyflavone) c cng thc cu to nh hnh 6.
H 3CO
8 7 9

O
4

6' 2 1'

5' 4'

OH

6 10 5

2'

3'

OH

OH

OH

Rhamnetin: C16H12O7
Hnh 6: Cng thc cu to ca LA6-9

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2.2 Kho st s b hot tnh khng oxi ha ca cc cht tinh khit c lp c

Bng 3: hp thu ca h phn ng gia cht tinh khit v DPPH

Tn cht

hp thu Ln o 1 Ln o 2 Ln o 3 Gi tr trung bnh Ac

LA2-2 LA6-9

0,211 0,211

0,211 0,211

0,211 0,211

0,211 0,211

Bng 4: Gi tr % c ch Q ca cc cao chit v cc cht tinh khit

K hiu LA2-2 LA6-9

% c ch Q 90,83% 90,83%

3 KT LUN Cc kt qu nghin cu trn y cho thy t C Lo Ph Yn c lp c thm hai flavonol thuc nhm hp cht flavonoid l rhamnocitrin (3,5,4-trihydroxy-7methoxyflavone) v rhamnetin (3,5,3,4-tetrahydroxy-7-methoxy-flavone). Hai cht ny c tnh khng oxi ha mnh tng ng nhau. TI LIU THAM KHO
Apichart Suksamrarn, Apinya Chotipong, Tananit Suavansri, Somnuk Boongird, Puntip Timsuksai, Saovaluk Vimuttipong and Aporn Chuaynugul, 2004. Antimycobacterial activity and cytotoxicity of flavonoids from the flowers of Chromolaena odorata. Arch Pharm Res, Vol. 27, No. 5, pages 507-511. David S. Seigler and E. Wollenweber, 1983. Chemical variation in Notholaena standley. Amer.J.Bot., 70 (5), pages 790-798. Maria Rose Jane R. Albuquerque et al., 2006. Terpenoids, Flavonoids and other constituents of Eupatorium betonicaeforme (Asteraceae). J. Braz. Chem. Soc., Vol. 17, No. 1, pages 68-72. Ng Quc Lun, Lm Thanh Phong, Nguyn Ngc Hnh, 2006. Mt s kt qu nghin cu thnh phn ha hc ca tinh du v flavonoid trong cy C Lo. Tp ch khoa hc, Trng i hc Cn Th, S 6-2006, trang 103-110. Ng Quc Lun, Nguyn Ngc Chu, Nguyn Ngc Hnh, 2007. Phn lp v nhn danh cu trc mt chalcone t dch chit ethyl acetateca cy C Lo- Eupatorium odoratum L. Tp ch khoa hc, Trng i hc Cn Th, S 8-2007, trang 16-20. Nguyn Th Ngc T, L Th Hi Yn, 2000. Nghin cu tc dng khng khun ca mt s thnh phn trong cao C Lo. Tp ch Dc hc, S 7, trang 17-19. R.N. Barua, R. P. Sharma, G. Thyagarajan, Werner Hertz, 1978. Flavonoids of Chromolaena odorata. Phytochemistry, Vol 17, pages 1807-1808. Toan Thang Phan, Lingzhi Wang, Patrick See, Renee Jacqueline Grayer, Sui Yung Chan and Seng Teik Lee, 2001. Phenolic Compounds of Chromolaena odorata Protect Cultured Skin Cells from Oxidative Damage: Implication for Cutaneous Wound healing. Biol. Pharm. Bull, Vol 24, issue 12, pages 1373-1379.

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