TAP CHI HOA HOC T. 49(3) 367-370 THANG 6 NAM 2011
AXIT SHIKIMIC VA CAC AXIT PHENOLIC PHAN LAP TU' V 6 CAY DE FICUS REUGIOSA L. (MORACEAE)
Cam Thj Inh', Tran Hong Quang^ Hoang Thanh Huong', Chau Van Minh^ Phan Van Kicm^
Vien Hda hpc Cdc hop chdt thien nhien, Vien Khoa hpc vd Cdng ngh? Vi^t Nam Vien Hda sinh bien, Vien Khoa hpc vd Cdng nghe Viet Nam
Den Tda soan 18-3-2011
Abstract
The ethyl acetate extract of the barks of Ficus religiosa L. showed moderate antioxidant activity in DPPH radical scavenging assay with SC50 value is 23.1 pg/ml. Five compounds including vanillic acid (1), 4-hydroxybenzoic acid (2), protocatechuic acid (3), gallic acid (4) and shikiinic acid (5) were isolated from the barks of this plant. Their structures were identified by Mass and NMR spectra.
l.MODAU
cay de Ficus religiosa L. (hg Moraceae) la mdt trong nhirng thuc vat dugc sir dung lau ddi nhat trong y hgc dan tdc An do va nhieu nudc de trj cac benh ngoai da (eczema, da lieu), tieu hda (da day, ly), tieu dudng va mdt sd benh lien quan den he than kinh trung uong [1 3].
Den nay da cd mdt sd cdng bd ve tac dung dugc ly [4, 5] nhung trudc chiing tdi hau nhu chua cd nghien cuu nao ve thanh phan hda hgc cua loai cay nay.
Gin day chiing tdi da thdng bao ket qua phan lap va xac dinh clu true hda hgc ciia 11 hgp chat trong dd cd hai hgp chit mdi la (3S,5R,6R,7E,9R)- megastiginan-7-en-3,5,6,9-terol-9-0-/?-
apiofuranosyl-( 1 "->2")-0-/?-D gluco-pyranoside, (3S,5R.6R,7E,9R)-inegastigman-7-en-3,5,6,9-terol- 9-0-y5-D gluco-pyranoside tii' djch chilt metanol la cay [6 9]. Tiep tuc tim bilu vl loai nay chung tdi da nghien ciru thanh phin hda hgc ciia vd cay. Bai bao nay cdng bo kit qua phan lap, xac djnh cau true hda hgc ciia axit shikimic va cac axit phenolic tir vd cay.
2. THUG NGHIEM 2.1. Phuong phap chung
; Dilm chay dugc do tren may Kofler micro-
^,hotstage. Bo quay cue dugc do tren may Polatronic D Schimidt + Haench. Pho khdi El-MS (Electron ioniption-Mass Spectra) dugc do tren may Hewlett
Packard 5989B MS Engine. Phd cdng hudng tir nhan (NMR) dugc do tren may Bruker AM500 FT-NMR Spectrometer.
sac ky ldp mdng dugc thuc hien tren ban mdng trang sin DC-Alufolien 60 F254 (Merck 1,05715), RP|8 F254S (Merck). Sac ky cdt dugc tien hanh vdi chat hap phu. Silica gel pha thudng cd cd hat 240 430 mesh va Silica gel pha dao ODS hoac YMC (30
50 pm, FuJisilisa Chemical Ltd.), Sephadex LH-20 (Pharmacia, Swenden).
2.2. Nguyen lieu
Vd cay F. religiosa dugc thu hai vao thang 9 nam 2007 tai Tam Dao, VTnh Phuc. Ten khoa hgc dugc TS Tran Huy Thai, Vien Sinh thai va Tai nguyen Sinh vat, Vien Khoa hgc va Cdng nghe Viet Nam giam djnh. Mau tieu ban dugc luu giu tai Vien Hoa hgc cac Hgp chat Thien nhien, Vien Khoa hgc va Cdng nghe Viet Nam. Nguyen lieu dugc xir ly diet men va say khd d nhiet do 60°C.
2.3. Chiet xuat va phan lap
200 g bdt vd khd F religiosa L. dugc chiet sieu am vdi metanol trong 12 gid (3 lan), sau khi loai dung mdi dudi ap suat giam, tien hanh chiet pha Idng-ldng vdi hai loai dung mdi nudc-etylaxetat (1:1). Phan djch chiet etylaxetat dugc sac ky tren cdt silicagel vdi he dung mdi gradien clorofoc-metanol (20:3) thu dugc 5 phan doan A, B, C, D va E. Tir phan doan B bang sac ky tren cdt Sephadex LH-20 va tinh che lai tren cdt silicagel nhan dugc cac hgp 367
TCHH, T. 49(3), 2011 Cdtn Thi Inh vd cdng sir chit 1 (7 mg) va 2 (13 mg). Slc ky nhIc lai phan
doan C cho hgp chat 3(18 mg). Tach phan doan D tren cdt pha dao va tinh chl tilp Ircn cgt silicagel pha thuan cho cac chit 4 (8 mg) va 5 (11 mg).
2.4. Hilng so vat ly vii du' lieu phd Hgp chat 1:
Tinh the hinh kim manh, dicm ndng chiiy 208 209T.
ELMS/;/-- 168 [M'].
'I l-NMR (500 MI Iz. CDCI3 + CD3OD) 6 (ppm):
7.65 (HI. dd. ./= 2,0. 8,5 11/, 11-6), 7,56 (111, d, ./ = 2.0 11/,. 11-2). 6,91 (111, d, .7=8,0 Hz, 11-5), 3,93 (311. s, 3-OCII3).
"C-NMR (125 MHz. CDCI3+ CD,OD) 6 (ppm):
169.1 (C-7), 150.5 (C-4). 146.5 (C-3), 124,5 (C-6), 121.8 (C-l), 114,3 (C-5), 112.3 (C-2), 55,9 (3-OCHO.
Hgp chat 2:
Dang bdt mau trang. Dicm ndng chay 213 214T.
EI-MS///r.' I38[M'].
' H - N M R (500 MHz, CDCI3+CD3OD) 6 (ppm):
7,91 (2H, d, J = 8.5 Hz. H-2 va H-6), 6,84 (2H, d, J
= 8.5 Hz, H-3 va H-5).
'-'C-NMR (125 MHz, CDCI3 + CD3OD) 6 ppm):
169.01 (C-7), 161.43 (C-4), 131,68 (C-2, C-6), 121,31 (C-1), va 114.81 (C-3 va C-5).
Hgp chat 3:
Dang tinh the mau vang. Dicm ndng chay 196 I98"C.
EI-MS m/z I54[M^].
'H-NMR (500 MHz, CDCI3) 5 (ppm): 7,51 (HI, d, J = L8 Hz. H-2), 7,49 (IH, dd, J = 1,8, 8,5 Hz, H-6), 6,85 (1H, d, 7 = 8,0 Hz, H-5).
"C-NMR (125 MHz, CDCI3) 6 (ppm): 169,3 (C-7), 150.9 (C-4), 145,5 (C-3), 123,8 (C-6), 123,1 (C-l), 117,3 (C-2), va 115,5 (C-5).
Hgp chat 4:
Tinh the hinh kim, trang due. Dicm ndng chay 235 237"C.
EI-MS/;//r: 170[M'].
COOH
'H-NMR (500 MHz, CD3OD) 5 (ppm): 7,08 (2H, s, H-2 va H-6).
'^C-NMR (125 MHz, CD3OD) 5 (ppm): 169,8 (C-7), 146,5 (C-3 va C-5), 137,8 (C-4), 121,4 (C-I), va 110,0 (C-2 va C-6).
Hgp chsit 5:
Dang tinh thi mau trlng [a]D -176° (c = 1,0, II2O).
Dilm ndng chay 185 186''C.
EI-MS////r: 174[M'].
'11-NMR (500 MHz, DMSO) 5 (ppm): 6,57 (IH, d,./= 1.5 llz, H-2), 4,02(111, s, H-3), 3,84(lH,dd, ./ = 10,5. 4,5 Hz, 11-5), 3,54(1 LI, dd, J = 5,5, 4,0 Hz,
11-4), 2,42 (111, ddt, J = 18,0, 5,0, 1,5 I Iz, H-6a), 2,01 (lII,ddt,./= 18,0,6,5, 1,5 Hz, H-6p).
'^C-NMR (125 MHz, DMSO) 5 (ppm); 168,07 (C=0), 138,86 (C-2), 128,45 (C-l), 70,43 (C-4), 66,91 (C-5), 65,58 (C-3). va 30,01 (C-6).
2.5. Hoat tinh chong oxi hda
Hoat tinh chdng oxi hda thdng qua phan iing bao vay gdc tu do la phuong phap danh gia kha nang tbu dgn gdc tu do theo phirong phap ciia Shela G. va cdng su [10]. Hoat tinh dugc danh gia thdng qua gia trj hap thu anh sang ciia dich thi nghiem so vdi doi chung khi dgc d may Elisad budc sdng 515 nm.
Mau can chiet etylaxetat vd cay F religiosa dugc pha theo 5 thang ndng do. Gia trj SC50 dugc xac djnh bang chuong trinh table curve thdng qua ndng dd chat thu va % hoat ddrn^ ciia chat thir ma d dd 50% cac gdc tu do tao bdi DPPl 1 dugc trung hoa bdi chat thir.
Ket qua SC50 la 23,1 pg/ml.
3. KET QUA VA THAO LUAN
Dich chiet etylaxetat vd cay F religiosa L. co boat tinh thau tdm gdc tu do kha manh vdi gia trj SC50 lil 23,1 pg/inl. ^^
Tir can chiet, bang nhung phuong phap sac ky thich hcTp chiing tdi da tach dugc nam hgp chat 1-5.
Cau tnic hda hgc ciia cac chat dugc xac djnh bang phuong phap phd cdng hudng tii' hat nhan va pho khdi lugng.
COOH
(1) (2) (3) (4)
R' OCH3
H OH OH
R-^
H H H OH
'OH
OH (5)
368
TCHH.T. 49(3). 2011 Axit shikimic vd cdc phenolic...
Cac hgp chat 1 5 cho mau xanh dam vdi dung djch FeCl3 dac trung cho nhdm phenolic. Du' lieu phd NMR (xem phan thuc nghiem) cho thiy cac phd
' T - N M R deu cd nhung tin hieu cua 7 cacbon lai hda sp', trong dd cd mdt tin hieu cua cacbon cacboxyl lien hgp tai 6c 169,1 169.X ppm vii mdt tin hieu cua cacbon bac 4 aromatic tai 5c 121,X
123,1 ppm. Dieu nav cbii'ng to bon chit niiv la nhii'ng axit phenolic dang C,,- Ci. Sir khac biet vl so lugng va ban chat cac nhdm the chira oxi o vdng benzen dan den su khac biet ve su chuyen dich hda hoc cua cacbon o' vdng thom (dac biet C-3. C-4 va C-5) cung nhu su khac biet ve sd lucing proton va khdi lugng phan tu cua cac chat.
Hai hgp chai 1 vii 3 co cac phd NMR kha gidng nhau, ciing cd tin hieu cua 2 cacbon aromatic lien ket vdi oxv tai {\ 146.5 va 150,5 ppm (o chat 1), (\
145,5 va 150,9 ppm (CT chat 2). Diem khac biet duv nhat chl o' chd tren cac phd NMR cua 1 cdn cd them tin hieu cua mdt nhdm metoxi tai cSc tai 55.9 ppm v ii 6H 3,91 (3H.S). Nbu vav. hgp chiu 1 chinh la dan xuat metoxi cua hgp chat 3. Dieu niiv cung hoan loan phii hgp v di su khac biet v e khdi lugng phan tu cua chung (khdi lugng phan tu cua chat 1 la 168 va cua chat 3 la 154).
So sanh hang sd vat ly. dir lieu phd vdi tai lieu tham khao [11, 12] da xac dinh dugc hgp chat 1 la axit vanillic v a hgp chat 3 la axit protocatechuic.
Hgp chat 2 cd pic ion phan tu mz 138. Ket hgp vdi diJ lieu phd NMR (xem phan thuc nghiem) xac dinh dugc cdng thirc phan tu la C-H^O?. Su xuat hien tin hieu cua cac cap proton aromatic tai 6H 7,91 (2H, d) va 6,84 ppm (2H. d) irng vdi phan tu cd ddi xirng true bac 2 cimg vdi tin hieu cua 1 cacbon aromatic lien ket vdi nhdm hydroxy tai 5c 161.43 ppm da chirng minh hgp chat 2 la axit 4-hydroxybenzaic. Dieu nay dugc xac nhan qua su so sanh hang sd vat ly va du' lieu phd cua chat 2 vdi tai lieu tham khao [13].
Hgp chit 4 cd pic ion phan tu m/z 170 irng vdi cdng thirc phan tu CTHCO,. Hang sd vat ly, cac du' Heu phd ciing su so sanh vdi tai lieu [13] va trg'c tiep vdi chit chuan cho phep khang dinh hgp chat 4 la axh gallic.
Hgp chit 5 cd pic ion phan tu m/z 174. Ket hgp vdi dij lieu phd NMR xac dinh dugc cdng thuc phan tulaC7H,,05.
Tren phd 'H-NMR cd tin hieu cua mdt proton metin a 6,57 ppm (d, J = 1.5 Hz), hai proton metylen 0 2.42 ppm (ddt.J= 1.5. 5,0. 18.0 Hz) va 2.01 ppm (ddL J= 1.5. 6.5. 18.0 Hz), cd ba proton hydroxyl metin a 4.02 ppm (s. H-3), 3,84 ppm (dd, J = 4,5,
10.5 Hz), va 3,54 ppm (dd. 7 = 4,0, 5,5 Hz).
Phd ' T - N M R cho bilt trong phan tu cd mat 7 nguyen tu cacbon, kit hgp vdi phd DEPT cho thay CO mdt cacbon cacbonyl (C=0) nlm o viing trudng
thap 5c 168,07ppm, mdt cacbon bac bdn vdi 5c 128,45 ppm. mdt nhdm cacbon metinlen a Oc 30.01 ppm, mdt cacbon metin a 6c 138.86 ppm, va ba cacbon hidroxyl metin o 6^ 70,43. 66,91 va 65.58 ppm. Til' ket qua phan tich phd cung nhu so sanh hang sd \i\[ ly, dii' lieu phd cua hgp chat 5 vdi tai lieu tham khao [14] va chat chuan, chiing tdi da xac djnh hgp chat 5 la axit shikimic.
4. KET LUAN
Dich chiet etylaxetat cua vo cay de F religiosa L. the hien boat tinh chdng oxi hda tdt tren he DPPH vdi gia tn SC.M, la 23,1 pg/ml. Tii' vo cay da phan lap va xiic djnh cau triic 5 hgp chat la axit vanillic (1).
axit 4-bydroxybenzoic (2). axit protocatechuic (3) axit galic (4) va axit shikiinic (5). Day la cdng bd dau lien ve sir cd mat cua cac hgp chat nay trong vo cay F religiosa L.
Loi cdm on: Cdng trinh duac hodn thdnh vdi sir tdi trg kinh phi eua de tdi nghien ciru ca ban - N.AFOSTED. md sd 104.01.31.09.
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369
TCHH. T. 49(3), 2011 Cam Thi Inh vd cdng su 11. Wu T. S. et al. Phytochemisiry. 36(3). 785 788 DVD. 1982-2005. CCR press.
(1994). 14. I larry Adame. Neil A. Bailey, Roger Bretde, Richard 12. Gutzei D. et al. Chromatoizraphy. 65. I 7 (2007). ^ '*'^^- ^^^'''y" Frederickson, Edwin Haslam, Fiona S.
Maebeath and Gareth M. Davies. Tetrahedron, Vol.
13. Chapman & Hall, Dictionary of natural products on 52(25) 8565 8580(1996)
Lien lu': Cam Thj Inh
Vien llo;i hgc ciic hgp cbiii thien nhicMi Vicn Khoa hgc v;i Cdng nghe Vict Nam
18 Hoang Qudc Viet. ( iiii (iiiiy. Ha Ndi Email: camtbiinlifa valioo.com
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