TP CH KHOA HC, i hc Hu, S 65, 2011 NGHIN CU PHN LP V NHN DNG CU TRC ALKALOID TRONG DCH CHIT T L VNG

NEM (ERYTHRINA ORIENTALIS L. FABACEAE) THA THIN HU

Trn Th Vn Thi, Trn Thanh Minh Trng i hc Khoa hc, i hc Hu

TM TT
Hm lng alkaloid ton phn trong l vng nem (Erythrina orientalis L. Fabaceae) Tha Thin Hu l 0,18% (so vi hm kh). pht hin 10 alkaloid trong cao l. Trong , 2 alkaloid c phn lp v nhn dng cu trc bng LC-MS, UV-VIS and 1H-NMR. Chng l erythrartine v erythrartine N- oxide. Nhng alkaloid ny cha tng c pht hin trong l vng nem (Erythrina orientalis L. Fabaceae) trc y.

1. M u Vng nem - Erythrina orientalis L. (tn gi khc Erythrina variegata L.) l mt loi vng mc hoang hoc trng ph bin Vit Nam. L Vng nem c cha nhiu alkaloid, t lu c nhn dn ta s dng n hoc sc ung cha bnh km n, mt ng, thn kinh suy nhc. Alkaloid ton phn trong l vng nem c tc dng khng khun, tr tim, gy ng, an thn [1]. Mt s c s y t hin ang s dng cao l Vng nem kt hp vi mt s v dc liu khc lm thuc [2, 3]. Trn th gii c mt s cng trnh nghin cu v alkaloid ca cc loi Vng nem khc n [4], Philippin [5], M [6], nhng ch tp trung vo alkaloid trong v thn cy, hoa v ht, m khng quan tm nhiu n alkaloid trong l. Chng ti nghin cu phn lp v nhn dng cu trc alkaloid trong l vng nem nhm gp phn nng cao gi tr s dng ca cc alkaloid trong dc liu ny. 2. Thc nghim 2.1. X l s b l vng nem v xc nh m L vng nem c thu hi mt s a im trong thnh ph Hu. Sau khi thu hi v, l c loi b cung, ra sch, kh ngoi khng kh, sau sy kh 60 0C trong 4 gi v bp vn thnh bt. Bt l c dng lm mu chit alkaloid. m ca mu c xc nh bng phng php khi lng [7]. 2.2. Chit xut alkaloid ton phn Bt l c lm m bng etanol 960, NH4OH 12,5% v chit ngm kit bng etanol 960. Dch chit c ct loi dung mi n cn, ha tan cn bng H2SO4 2%, lc ly dch
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lc (cc alkaloid lc ny dng mui, khng tan c trong CHCl3). em dch lc loi tp bng CHCl3, ri a v pH = 9-10 bng NH4OH m c (cc alkaloid lc ny dng base t do, tan c trong CHCl3) v chit ly alkaloid bng CHCl3. Lc dch chit CHCl3 qua giy lc c Na2SO4 khan, ra giy lc v Na2SO4 khan bng CHCl3, ri bc hi CHCl3 (c ct 50 0C) thu c cn alkaloid ton phn [1, 7, 8]. 2.3. nh lng alkaloid ton phn tinh khit nh lng alkaloid ton phn tinh khit trong cn alkaloid ton phn bng phng php chun acid base [1]. 2.4. Phn lp cc cu t alkaloid S dng cc phng php: chit phn on trong cc mi trng pH khc nhau, chit phn on bng cc dung mi c phn cc khc nhau, sc k ct v sc k lp mng phn lp cc cu t alkaloid t cn alkaloid ton phn. - Chit phn on trong cc mi trng pH khc nhau: cn alkaloid ton phn c ha tan trong H2SO4 2%, sau ln lt chuyn ln cc phn on pH = 2, pH = 6 v pH = 9 bng dung dch NH4OH m c v chit bng chloroform [1, 8]. - Chit phn on cn alkaloid ton phn bng cc dung mi c phn cc khc nhau theo phng php chit rn - lng, nh tnh bng thuc th Dragendorff. - Sc k ct: ct sc k c nhi theo phng php nhi t vi ng knh 2 cm v chiu cao 20 cm. Cht hp ph l silica gel (Merck, c ht 0,063 0,200 mm), ra gii ln lt bng cc dung mi v h dung mi c phn cc tng dn. - Sc k bn mng: s dng bn mng nhm silica gel GF254 trng sn ca Merck, dy 0,25 mm. Trin khai vi h dung mi CHCl3 : CH3OH : NH4OH (50 : 9 : 1 theo th tch). Thuc hin mu l hi iod hoc thuc th Dragendorff. 2.5. Xc nh cu to ca alkaloid - Xc nh tinh khit v nh tnh bng sc k lng - khi ph LC-MSDTrap-SL (Agilent); iu kin sc k: dung mi methanol - nc (85:15), thi gian 35 pht, tc dng 1 ml/pht, ct ODS-C18, detector DAD, bc sng 284 nm; iu kin ghi ph khi: Ngun ion EFI, kh lm kh N2 (nhit kh 350 0C, p sut 30 psi, lu lng kh 15 lt/pht). Chn khng 2,5.10-5 mmbar. in th (nng lng detector) 1118 V. - nh tnh cu to bng ph MS, ph 1H-NMR Brucker Avance 500 MHz, dung mi DMS; ph m phng c xy dng trn phn mm Chemdraw Ultra 8.0 (ChemOffice), ph UV-VIS.

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3. Kt qu v tho lun 3.1. Hm lng alkaloid ton phn v s cu t alkaloid trong l Vng nem T 1604 g nguyn liu l Vng nem kh ( m trung bnh 9,97%), bng phng php chit ngm kit, sau khi tinh ch thu c 4,05 g cn alkaloid ton phn vi tinh khit 70,02% theo phng php chun acid - base, t l alkaloid tinh khit so vi hm kh l 0,18%. Cc gi tr Rf ca cc cu t trong alkaloid ton phn t sc k c trnh by trn ct (2), bng 1 cho thy cn alkaloid ton phn c cha 10 cu t trong gii hn pht hin ca sc k bn mng. 3.2. Phn lp cc cu t alkaloid Kt qu chit cc phn on alkaloid pH khc nhau, sau chit bng cloroform cho thy rng cc cu t alkaloid trong l vng nem u c tnh base yu, khng to mui pH=2. Tin hnh chit phn on cn alkaloid ton phn ln lt bng cc dung mi c phn cc tng dn t n - hexan n methanol, kt qu th hin trn ct (3), bng 1. Nh vy, cc alkaloid trong l Vng nem c phn cc rt gn nhau, do khng th phn lp bng cc dung mi tinh khit. Bng 1. Mt s tnh cht ca cc alkaloid thnh phn trong dch chit ca l Vng nem (SKBM: sc k bn mng, SKC: sc k ct) (1) STT 1 2 3 4 5 6 7 8 9 10 (2) Rf (SKBM) 0,84 0,77 0,70 0,61 0,56 0,49 0,43 0,38 0,31 0,23 (3) Chit ln trong dung mi n- hexane benzene, chloroform chloroform : methanol benzene, chloroform (5,5 :1) chloroform chloroform benzene chloroform chloroform chloroform chloroform
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(4) Phn on ra gii (SKC)

methanol

Bng sc k ct kt hp vi sc k bn mng iu ch, phn lp v tinh ch c hai cu t c Rf = 0,70 (3,0 mg) v Rf = 0,31 (1,4 mg). 3.3. Nhn dng cu trc ca cc alkaloid phn lp Cu t Rf (SKBM) = 0,70: tinh th hnh kim, mu vng nht, thi gian lu trn LC l 12,13 pht, ph MS c cc mnh ion: 330,0 ([M+H]+), 283,1; 192,1; 127,0 v 113,1; ph UV VIS c cc cc i hp th: 285; 255 v 230 nm. Cu t Rf (SKBM) = 0,31: tinh th hnh kim, mu vng nht, thi gian lu trn LC l 12,60 pht, ph MS c cc mnh ion: 346,0 ([M+H]+), 330,1 v 316,1; ph UV VIS c cc cc i hp th: 285 v 230 nm. T kt qu LC-MS cho thy, cu t Rf = 0,70 tng ng vi alkaloid erythrartine (C19H23NO4) v cu t Rf = 0,31 chnh l alkaloid erythrartine N-oxide (C19H23NO5). Cc Erythrina alkaloid c ghi nhn trn LC nh cht chun ni l cc alkaloid cng dy dienoid Erythrina hay alkenoid Erythrina. Mt khc, ph khi ng vai tr cc k quan trng trong vic xc nh cc Erythrina alkaloid [9]. Mi phn tch chi tit ph khi ca mt lot cc Erythrina alkaloid chng t rng, mnh u tin ca cc alkaloid dng dienoid v lactonic tng ng vi s b gy nhm metyl hay nhm metoxy t nhm metoxy ti C3. Cc alkaloid dng alkenoid phn mnh theo mt cch khc, nhng khuynh hng chung u l to thnh h lin hp di hn. Kt qu phn mnh ph khi ca cu t m chng ti tch c (hnh 1 v hnh 2) l hon ton ph hp vi c ch phn mnh ca cc Erythrina alkaloid c cu trc dienoid [9].
OH CH3O CH3O CH3O N -e CH3O CH3O CH3O N OH + H CH3O CH3O CH3O N + O H

erythrartine

m/z = 329

m/z 330
CH3O CH3O

-O -OCH3

m/z = 283
Hnh 1. S phn mnh ca cu t erythratine

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Cc Erythrina alkaloid thu c u c s mnh ion rt t do to h lin hp bn. iu ny ph hp vi ti liu [9]. Ngoi ra, ph UV-VIS ca cc cu t ny c cc cc i hp th ti cc bc sng max = 285 nm; 230 nm, l cc cc i hp th ca nhn thm c cha 2 nhm metoxy v h dien lin hp [5]. Ph 1H-NMR ca erythrartine N- oxide c cc peak ng vi (ppm): 8,50 (d) ca =CH-; 6,80 7,00 (s, s) ca =CH benzen; 6,70 (d) ca =CH-, 6,60 (s) ca =CH-; 5,80 (s) ca -CH- bc 3; 4,10 (s) ca OCH3; 3,82 (s) ca OCH3; 3,70 (d) ca CH2-; 3,50 (s) ca -OCH3; 3,40 (s) ca CH- bc 3; 3,20 3,40 (s, s) ca cc CH2-; 3,30 (s) ca -OH; 2,50(d) ca CH2-; 2,45 (d) ca CH2- hon ton ph hp vi ph m phng v c chng ti quy kt ph hp vi mi hydro ti cc v tr trong cng thc cu to ca erythrartine N- oxide.
OH CH3O CH3O CH3O + N O +
+H

OH CH3O CH3O H CH2 CH3O CH3O N+ -CH2 CH3O CH3O

OH

-O

m/z = 345

m/z = 330

m/z = 316

Hnh 2. S phn mnh ca cu t erythrartine N- oxide

Dienoid Erythrina alkaloid erythrartine v erythrartine N- oxide c pht hin trong hoa ca loi E. mulungu ca Philippin [5]. Ngoi ra, cu t erythrartine Noxide c pht hin t dch chit ht ca loi E. berteroana ca M bng phng php GC/MS [6], nhng cha c cng trnh no xc nh chng c mt trong l ca loi Vng nem Erythrina orientalis L. (hay Erythrina variegata L.) Vit Nam [10]. 4. Kt lun - Hm lng alkaloid ton phn trong l vng nem l 0,18% (so vi hm kh). Trong cn alkaloid ton phn c 10 cu t trong gii hn pht hin ca sc k bn mng. Cc cu t alkaloid trong l vng nem u c tnh base yu v c phn cc gn nhau. - phn lp c 2 cu t c Rf = 0,70 (3,0 mg) l erythrartine v Rf = 0,31 (1,4 mg) l erythrartine N- oxide. Hai cu t ny c pht hin trong hoa ca loi E. mulungu, Philippin v trong ht ca loi E. berteroana, M, nhng cha c cng trnh no xc nh chng c mt trong l ca loi Vng nem Erythrina orientalis L. (hay Erythrina variegata L.) Vit Nam.

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TI LIU THAM KHO

[1]. Nguyn Thanh K, Nguyn Th Tm, Trn Vn Thanh, Bi ging dc liu, Nxb. Y hc, 2002. [2]. Dc phm OPC, viewid=35&idloai=9&viewopt=text. [3]. Dc phm TW 5 product&dtd=sub&id=185. http://www.opcpharma.com/vn/viewchitiet.php? http://www.ecvina.com/danapha/?do=

Danapha,

[4]. Ghosal S., Dutta S. K., Bhattacharya S. K., Erythrina Chemical and Pharmacological Evaluation II: Alkaloids of Erythrina variegata L. J. of Pharmaceutical Sciences, Vol. 61(8), (1972), 1274 1277. [5]. Sarragiotto M. E. , Filho H. L., Marsaiolo A., Erysotrine-N-oxide and erythrartine-Noxide, two novel alkaloids from Erythrina mulungu, Can. J. Chem., 59, (1981), 2771 2775. [6]. Soto-Hernandez M., Jackson Anthony H., Erythrina Alkaloids: Isolation and Characterisation of Alkaloids from Seven Erythrina Species, Planta Med., 60, (1994), 175 177. [7]. B Y t, Dc in Vit Nam III, Nxb. Y hc, 2002, 503-504. [8]. Nguyn Vn n, Nguyn Vit Tu, Phng php nghin cu ha hc cy thuc, Nxb. Y hc, 1985. [9]. Chawla A. S., Kapoor V. K., Erythrina alkaloids, Handbook of Plant and Fungal Toxicants, 1997, 37 46. [10]. Arthur W., Craig R. www.traditionaltree.org, 2004. Elevitch, Erythrina variegata (coral tree),

STUDY ON ISOLATION AND STRUCTURE IDENTIFICATION OF ALKALOIDS IN FOLIUM EXTRACT OF ERYTHRINA ORIENTALIS L. FABACEAE COLLECTED IN THUA THIEN HUE
Tran Thi Van Thi, Tran Thanh Minh College of Sciences, Hue University

SUMMARY
The content of alkaloids of dry weight is 0,18% in folium of Erythrina orientalis L. Fabaceae collected in Thua Thien Hue. There were ten alkaloids found in folium extract. Two of them were isolated and identified by LC-MS, UV-VIS and 1H-NMR. They were erythrartine and erythrartine N- oxide. These alkaloids have not been defined in folium of Erythrina orientalis L. Fabaceae in the past. 230