» Nghidn cii-u •- Ky thujit
3. Gianinazzi S. and Glaninazzi-Pearson V.
(1968). "Progress and hsadaches in endomycon'hizai biotechnology", Symbk33ls, 2, pp, 139-49.
4. Ukhltayawuld K., Angerhofer C. K. (1993),
"Cytotoxic and antimalarial bisbenzyllsoqulnollne alkaloids from Saphania evecta". Jounal of Natural Products. 56 (1), pp. 30-38.
5. Shela O., Olga M. B„ Elen K. et al. (2003),
"Comparison of the contents of the main btochemk»l compounds and the antioxidant activity of some Spanish olive oils as determined by four different radical scavenging tests", Joumal of Nutdttonal Btochemistry, 14, pp. 154-159.
6. Vanden B. D. A., VlietJinck A. J. (1991), 'Screening methods tor antlbacteilal and antiviral agents from hight plants'. MetfKxfe in Plant BioOiemlstry. 6, pp. 47-68
f Mr
Hop chat flavonoid phan lap tir re cay den kim {Bidens pilosa L.,)
Phgm Vfin Vu^iig', Ph9in Thanh K^^, Nguyen Th| BIch Thu^ Phan Vfln Kifm*
' B?nh Vi4n Quan Y17- Quan Khu 5.
' Tntang Dgi hpe Dugc Ha Npi, ^Vi^n Dirge lifu
Vi^n Hoa sinh Bien - Vi^n Khoa hpe vd Cong nghe ^i?t Nam.O ^ t v a n d e
c a y don kim ed tdn khoa hyc Bidens pilosa L , thude hy Ciie- Asteraeeae, Ici cSy mye hoang d nhifeu noi tir mi^n nui, trung du, d i n g bSng v ^ nhieu n u d e trdn th6 gidi nhu: Trung Qudc, HSn Quoc, Nh^t BSn, Brazil . w D3y Id lodi cdy mpc quanh nSm. sire sinh sdn nhanh vi d u y c coi nhu loai "cd dgi"*''.
Nghien a>u vd thdnh phSn hda hpe cho th^y edy do-n kim cd chira flavonoid. Nhiki hyp eh4t flavonoid eua edy ndy da duyc phdn l$p vd chirng minh ed tae dpng chdng oxi hda. bdo vd gan ^- ^ ' .
Trong nghidn ciru trude, ehiing tdi da edng bd vd ede flavonoid phSn l^p tir ph^n trdn m$t dat cay do-n kim'^'. Bdi bdo ndy chiing tdi trinh bdy kdt qud phan l§p va xdc djnh cAu triie cua 2 h y p chat flavonoid tie re eay don kim.
N g u y e n l i e u v a p h u ' a n g p h d p n g h i e n c i > u
Nguyen l i ^ u
Re eay don kim d u y e thu hai tgi Yen Lge - VTnh Phiie vdo thdng 9 - 1 1 n§m 2010. M i u nghien c u u da d u y e PGS.TS Vu Xudn Phuang vd ThS. Do The C u d n g (Phdng thyc v | t - Vi^n sinh thai va Tdi nguyen smh vgt - Vign Khoa hoc va Cdng nghe Vipt Nam) xae dinh ten khoa hoe la Bidens pilosa L , thupe ho COe -
Asteraeeae. M l u tidu bdn d u y c tuu tgi Vi$n Sinh thdi vd Tdi nguydn sinh v$t.
P h i r o n g p h d p n g h i d n ci>u
- Chi^t xudt cde chdt trong d u y c li$u bing p h u o n g phdp ngdm vdi methanol d nhidt dO phdng.
- Phdn \ip ede ehdt bdng sde ky edt diing ehdt hdp php Id silicagel pha t h u d n g c d hgt Id 0.04-0,063mm (240 - 430 mesh. Merck) vd pha ddo ODS ho$e YMC (30-50 ^ m . Fujisilisa Chemical Ltd).
- s d c ky ldp mdng d u y c t h y c hi$n tren bdn mdng trdng sdn silica gel DC-Alufdien 60 Pm (Merck 1,05715), RP.e Fjs*, (Merck). Phdt hi§n chdt bdng ddn tu- ngogi d hai b u d c sdng 254 vd 365 nm, ho$e diing thu6c thif Id dung djch H2SO4 10% d u y c phun ddu ldn bdn mdng, sdy khd r6i ho ndng trdn bdp didn tir tir ddn khi hi§n mdu.
- Ph6 cdng h u ^ n g tir nhdn (NMR): ^H-NMR (500 MHz) vd ' ^ O N M R (125 MHz) do tren mdy Bmker AM500 FT - NMR Spectrometer, d V i ^ Hod hpe, Vign Khoa hyc vd Cdng nghd Viet Nam.
- Md td quy trinh chiet xudt vd phdn \ip cic h y p ehdt:
Re cdy d o n kim sau thu hai d u y c n>a sgeh, thdi nhd, phoi khd v d say d SO^C sau do nghidn nhd thu d u y c 3,0 kg bdt thd. Bdt ndy duyc
TAP CHi DU(?C HQC - 09/2012 (S6 437 NAM 52)
• Nghien CLPU - Ky thugt
ngdm chidt 3 ldn, ldn ddu 48 g i d , 2 ldn sau 24 gid, mdi ldn diing 10 lit methanol. Gdp cdc djch chidt, cdt thu hoi dung mdi d u d i dp sudt gidm thu d u y c 158,0 gam djeh ed d$m d $ c Dieh ndy d u y c phdn tdn vdo 2 lit nude cdt, tidn hdnh ehidt phan b6 Idn l u y t vdi clorofonn (3 x 1,5 lit), ethyl acetat (5 x 1,5 lit), n-butanoi (5 x 1,5 lit).
Cde djch ehidt clorofomi, ethyl acetat, n-butanol d u y e edt thu h6i dung mdi d u d i dp sudt gidm ddn khi ki$t dung mdi thu d u y e ede cdn theo thip t y : cdn P, (55,0 g), cdn P; (25,0 g), edn Pg (28,0 g) vd Idp nude edn Igi.
Tir can P2, phdn l$p trdn cdt silicagel pha thudng vdi hd dung mdi CHCI3/ MeOH vdi dd phdn eye tdng ddn (90:10. 85:15. 80:20, 75:25, 70:30). Sau khi logi dung mdi d u d i dp sudt gidm d u y e 5 phdn dogn tuong ung Id P2A ddn P2E.
Phdn dogn P2E tidp tgc d u y e phdn l§p trdn cdt sir dyng slicagel pha thudng vd pha ddo vdi hg dung mdi rira gidi thich h y p d u y e hyp ehdt BP8 d u d i dgng tinh thd mdu vdng chanh (18mg).
Hyp ehat BP7 (27 mg) d u y e phdn lap tir phan dogn P2C sir dyng cpt silicagel pha thudng vd pha dao vdi hd dung mdi rCra giai thich hyp.
Ket qua va thao luan
Hyp chdt BPT nh^n d u y c d u d i dgng bdt mau vang dac trung cua mpt flavon. Cde pho NMR eua hop ehat ndy cho thay day Id mot flavon-glyeosid vdi nhanh dudng id a-L- arabinofuranosyl.
Phd ^H-NMR eiia BP7 xuat hien tin hi$u eua hai proton eua vdng thom bj the bon vj tri tai 6H 6,20 ( I H , d, J = 2,0 Hz) va 5H 6,40 ( I N , d, J = 2.0 Hz), ba proton eiia vdng thom bj the meta va para tgi 5H 6,84 (1H, d, J = 8,0 Hz), 7,48 (1H, d, J = 2,0 Hz) va 5H 7,56 ( I H , 6, J = 8,0, 2,0 Hz) cdc d u kipn trdn cho phep d u dodn BP7 Id mpt h y p chat flavonoid ed khung dgng quereetin.
Ngoai ra, ti'n hieu eua mdt proton anomer cung d u y c xae djnh tai 5H 5,58 ( I H , s). Chii y ddn hang sd t u o n g tde J eua proton anomer eiia phan t u dudng cd the thay rdng proton H-1" (5 5,58) ed gid trj JH-I"/H-2'' = 0 Hz ehung td d dd hinh thanh lien ket a- arabinofuranosid vd eae tin hieu eua eae nhdm methin gan vdi nhdm hydroxyl xuat hi§n d vung 5H 3,28 - 4,15 ppm.
TAP CHI DlTgfC HQC - 09/2012 (SO 437 NAM 52)
BAng 1 : D u liiu phd /VMR cCia hpp chit BP7 VI tri
C 2 3 4 5 6 7
e
9 10
r
2' 3' 4' 6' 6' 1"
2"
3"
4"
5"
V ™
157,4 133,8 178,0 161,3 99.1 164,5 94.1 156,8 104.3 121.4 115.8 145.3 148,7 115,9 122,2 108,2 82.3 77.2 86.6 60.9
&••
156.86 133.34 177,65 161,16 98,59 164,14 93,48 156,29 103.91 120.91 115.49 145.01 148,39 115,49 121,62 107,81 82.05 76.93 85.83 60.63
mult. (J In HI
6,20 d (2,0) 6.40 d (2.0)
7,48 d (2.0)
6.84 d (8.0) 7.56 dd (2,0,
8,0) 5,58 s 4,15 bis 3,72 brs 3.56*
3.28' HMBC (H-fC)
5. 7. 8. 10 6. 7, 9, 10
2.3'
r. 3' 2, 2'. 4'. 5'
3, 2", 3".
4" 4" 2", 4". 1"
3" 4"
^ measured in DMSO; "75 MHz; "300 MHz; ^125 MM; '500 MHz. * overia;
'dc^'" measured in DMSO
Phd " c - N M R ciia BP7 xuat hign tin higu eiia 20 carbon, trong dd ed mpt nhdm metilen tgi 5c 60,63 ppm, chin nhdm methyl tir 6c 76.93 ppm ddn 5c 121,62 ppm. chin nguyen tii' carbon bge 4 tCr 6c 103,91 ppm den 5c 164,14 ppm va mdt nhdm carbonyl tgi 5c 177,65 ppm. Phdn tich ky ede tin hidu tren pho ^^C-NMR cua BP7 eho thay, 15 nguyen t d carbon thupe khung quereetin, 5 nguyen tCr carbon cdn Igi thudc vdo 1 phdn t u dudng '^'.
Ha OH Hinh 1 : Ciu true hda hoc cua hop chat BP7
• Nghlfin cii-u - Ky thujit
Bi x i c djnh chinh x i c v| tri c i c liSn k^t cGng nhu- c^u trOc cOa phdn tir 6\^nQ, di Hin h i n h ao phft hai c h l i u HSQC vi HMBC, P h i n tich c i c tirang t i c t r i n p h i HSQC v i HMBC m$t \kr\
nOa khdng djnh phdn aglycon cOa hep chdt BP7 c6 khung quereetin v M s i / xudt hidn cCia tu'ong t i c HMBC g » a H-6 (6H 6.20) vdl C-5, C-7, C-8 v i C-10; gl&a H-8 (BH 6.40) v * l C-6. C-7, C-9 v i C-10; glQ'a H-2' (6„ 7,48) v « C-2. C-3'. g » a H-5' (B„ 6,84) v * l C-1', C-3' v i glOa H-6' (6„ 7,56) v * l C-2, C-2', C-4' v i C-5'. N g o i l ra, tirang t i c gi&a proton anome cua p h i n tt> durirng tai 6H 5.58 v * l cartion C-3 (6c 133.34) <]i x i c djnh vj tri l i i n kdt cua gdc du'dng n i y tai cartxin C-3. C i c ti/ong t i c cua H-1" v * i C-3". C-4"; H-2" v * l C- 3". C-4", H-3" vfti C-2", C-4", C-5". H-4" viirl C-3"
v i H-5" viW C-3". C-4" x i c djnh vj tri cua c i c cartion cua p h i n tu* dird^ng.
, 0 H
HO" ' OH Hinh 2: Cic tuxyng tic HMBC chinh cua hpp chit BPT
P h i n tich dO djch chuydn h o i hoc v i c i c t u a n g t i c t r i n phd cOng hu-dng tCr hat n h i n v i so s i n h V ^ I t i i ll$u tham khio'^', cdu trOc cua gdc 6[sirng iirqrc x i c djnh l i arabinofuranosid, v i hop chdt BP7 du-pc x i c djnh l i quercetln-3- a-L-arabinofuranosid vO\ cong thuc p h i n tur l i CzoHieOn D i y l i mit hi?p chit lin diu tiin dw<?c phin lip tir ri toil Bidens pikrsa v i da d u a c p h i n l i p t u i o i i Bidens sulphured.
Hap chdt BP8 dL^ac p h i n lap 6* p h i n doan ethyl ecetat, dgng tinh the m i u v i n g d i e tryng cua mot flavon. C i c phd NMR cua hap chdt n i y cho thay day l i mpt flavon-glycosid t u a n g t y nhu hop chat BP7
Phd ' H - N M R cua BP8 xudt h i i n tin h i i u hai proton cua vong tham bj the bon vj tri tai 6H 6,20 ( I H , d. J = 2,0 Hz) v i B„ 6,40 ( I H , d, J = 2,0 Hz), ba proton cua vong tham bj thd meta v i para tai 5H 6,80 ( I H , d. J = 8.0 Hz). 7,52 ( I H . d.
J = 2,0 Hz) v i BH 7,66 ( I H . d. J = 8.0, 2,0 Hz)
c i c dO' k l j n t r i n cho p h i p d y d o i n BP8 l i mOt h e p chdt (lavondd c 6 khung dang quereetin.
N g o i l ra, tin hl^u cila mOt proton anomer cOng 6uKfC x i c dinh tai 6H 5,36 (1H, d , J = 7,5 Hz).
ChCi i ddn hdng sd tuwng t i c J o i a proton anomer cCia p h i n ti> d u & n g c6 thd thdy rdnj proton H - 1 " (6 5,36) cd g i i tri JH-I-VH-Z- = 7,5 Hz chi>ng td d i hinh t h i n h l l i n kdt ^ y o o s l d vS c i c tfn hidu cOa c i c nhdm methin gdn vd'i nhdm hydroxyl xudt hidn ir viing 6H 3,32 - 3.65 ppm.
Bing 2: DCrlliu phi NMR cOa hop chit BP!
VI trie 2 3 4 5 6 7 6 9 10
r
2' 3' 4' 5' 6' 1"
2"
3- 4"
5"
6"
V'ra
156,5 133,7 177,6 161,1 gs,s 164,2 93,7 156,4 104,0 121,3 116,1 144,8 146,4 115,3 122,0 102,0 71,3 73,2 67.9 75.9 60.2
* "
156.28 133.50 177.48 161.20 98.64 164.11 93.47 156.23 103.90 121.09 115.96 144.78 148.42 115.16 121.94 101,85 71,20 73,19 67.90 75.80 60.12
mult. (J In Hz)
6.20 d (2.0) 6.40 d (2,0)
- -
7.52 d (2.0)
-
6.80 d (8.0) 7.66 dd (2.0.8.0)
5.36 d (7.5) 3.571(8,5)
3.36 bis 3.65 bre 3.32- 3.37-
" do trcng DMSO. "125 KtHz, '500 MHz; • overtsf;
'Sc do tmng DMSO
Phd-"C-NMR cua BP8 xudt hi$n tin h i i u cOa 21 cartxjn, trong dd c6 mOt nhdm methllen tai 5c 60.12 ppm, m u 6 i nhdm methin til 5c 67,90 ppm ddn 6c 121,94 ppm. chin n g u y i n tiJ carbon bic 4 t i l 6c 103.90 ppm ddn 6c 164,11 ppm v i mdt nhdm carbonyi tai 6c 177.46 ppm. Phan tich ky c i c tin hiSu t r i n phd " C - N M R cua BP8 v i so s i n h v d l hap c h i t BPT cho thdy. cung gidng nhu- BP7, BP8 c6 15 nguyen ti> cartwn thuOc khung quereetin nhung BP8 cd 6 nguyin ti>
carbon cdn iai thugc v i o 1 phan tO d u d n g trong khi BP7 lal l i d u * n g 5 carbon'".
36 TAP CHl Dl/giC HQC - 09/2012 (S6 437 NAM 52)
• Nghien CIFU - Ky thugt
Phdn tich dd djch c h u y i n hod hpe trdn ph6 cOng h u d n g ti> hgt nhdn cCia BP8 vd so sdnh vdi ehdt s6 1 trong tdi li$u tham khdo'^', c4u trdc eOa g6c d u d n g dup'c xde ^ n h Id galactopyranosid, vd hp'p chSt BP8 dup'c xdc djnh Id quercetln-3-^-D-galactopyranosid vdi cdng thdc phdn tCr Id C2iH2oOi2- Bdy cung Id mdt hp'p chSt \in 6iu tidn dup'c phdn l$p t u r§
cdy don kim vd cung da dup'c phdn l$p t u lodi Bidens sulphurea.
^ ^ . O H
HO
Hinh 3: Ciu true hda hpe cua hop chit BP8 K e t l u a n
B i n g ede phuong phdp sde ky k^t hp'p, cdc hp'p c h i t quereetin-3-a-/.-arabinofuranosid (BP7), quereetin-3-iS-D-galaetopyranosid (BP8) da duoc phan Igp tCr djch chiet methanol cua r§ cay don kim. Cau tnJc hda hoc cua cac hp'p chat nay duoc xac djnh bdng phuang phdp pho epng hudng t u hat nhdn (NMR) vd ddi chi^u vdi tdi lieu tham khao. Ddy la 2 hop chat flavonoid l i n dau ti6n cdng bd dup'c phan lap t u rd cay den kim.
S u m m a r y
Two natural flavonoids were isolated from
methanol extracts of the roots of Bidens pilosa L (Asteraeeae) by combination of various chromatography techniques. They were Identified as quercetin-3-a-L-arabinofuranoside (BPT) and quercetin-3-p-D-galactopyranoside (BP8) by NMR Including ID- and 2D-NMR. Both flavonoids were isolated for the first time from the roots of Bidens pilosa L
Keywords: Bidens pilosa L., flavonoids. root.
T d i l i ^ u t h a m k h a o 1 Phgm V3n Vupng, Phgm Thanh Ky, Hodng van Luang. Phan Vdn Ki$m (2010), "Cdc hpp ch4t flavonoid chi4t xudt tu cdy dan kim {Bidens pibsa L )", T^p chl Dupe hQC s6 409, th^ng 5, trang 48-53.
2. Chiang, Y. M., et al. "Metabolite profiling and chemopreventive bioactivity of plant extracts from Bklens pihsa." (2004) Jour of Ethnophaiwacol.;
95(2-3): 409-19
3. Edilene Delphino Rodrigues, Denlse Brenta da Silva, Diondia Camilo Rodrigues de Otiveira and Gil Valdo Jos6 da Silva (2009), "DOSY NMR applied to analysis of flavonoid glycosides from Bidens sulphurea", Magnetic resonance in chemistry, , Vol.
47,1095-1100.
4 Wang, J., Yang, H, Lin. ZW., Sun, H.D..
(1997), "Flavonoids from Bidens pilosa var. radiata", Phytochemistry 46: 1275-1278.
5. Wang R, Wu QX, Shi YP (2010), "Poiyacetylens and flavonoid from the Aenal Parts of Bidens pibsa".
Planta Med. 76. 893-896
6. Yuan L.P., Chen F.H., Ling L.. Dou P.F , Bo H., Zhong M.M., Xia L.J., (2008), "Protective effects of total flavonoids of Bidens pilosa L. (TFB) on animal liver Injury and liver fibrosis", Jour, of Ethnopharmacol., 116: p 53^-546.