B Y T

TRNG AI HOC DC HA NI

**********

NGUYN TIN TIP

NGHIN CU THANH PHN HOA

HOC LA U U RNG
(Trevesia palmata (Roxb. & Lindl.) Vis.)

KHOA LUN TT NGHIP DC S

HA NI 2014

B Y T
TRNG AI HOC DC HA NI

**********

NGUYN TIN TIP

NGHIN CU THANH PHN HOA

HOC LA U U RNG
(Trevesia palmata (Roxb. & Lindl.) Vis.)
KHOA LUN TT NGHIP DC S
Ngi hng dn:
TS. Quyn
Ni thc hin:
B mn Dc liu
Trng ai hoc Dc Ha Ni

HA NI 2014

LI CAM N
Trong qu trnh thc hin kha lun tt nghip, ti nhn c nhiu s
hng dn v gip ca thy c, gia nh v bn b.
hon thnh kha lun ny, trc ht ti xin by t s knh trng v lng
bit n su sc ti TS. Quyn, ngi thy giu lng am m, lun tn tnh
hng dn, ng vin, truyn t cho ti rt nhiu kin thc v nim am m trong
cng vic.
Ti xin by t s knh trng v lng bit n su sc ti ThS. Phm Tun
Anh, ngi thy tn tnh hng dn v truyn t cho ti nhng kin thc cng
nh nhng kinh nghim nghin cu khoa hc qu bu. Thy l tm gng sng
trong nghin cu. Phong cch lm vic ca thy to cho ti nim am m ln.
Ti cng xin by t lng bit n su sc ti TS. Nguyn Qunh Chi v Th.S.
L Thanh Bnh hai c gio lun nhit tnh v tn tm vi nghin cu. Nhng h
tr qu bu ca hai c gip ti hon thnh kha lun ny.
Ti xin chn thnh cm n cc thy c, cc anh ch k thut vin ang cng
tc ti b mn Dc Liu Trng i hc Dc H Ni to iu kin thun li
ti hon thnh kha lun.
Ti cng xin trn trng cm n tt c cc thy c gio trng i hc Dc
H Ni dy bo, truyn t kin thc cho ti trong nhng nm hc va qua.
Cui cng, cho php ti by t lng bit n v hn ti gia nh, bn b nhng
ngi lun quan tm, ng vin v l ch da tinh thn vng chc cho ti hon
thnh tt kha lun ny.
H Ni, ngy 14 thng 5 nm 2014
Sinh vin
Nguyn Tin Tip

MUC LUC
Trang
T VN ............................................................................................................1
CHNG 1. TNG QUAN .....................................................................................2
1.1. CHI Trevesia Vis. V LOI Trevesia palmata (Roxb. & Lindl.) Vis. ........2
1.1.1. c im thc vt .....................................................................................2
1.1.2. Thanh phn hoa hoc ca chi Trevesia Vis. va cy u rng ............7
1.1.3. Tac dung va cng dung ca cy u rng .........................................8
1.2. TRITERPENOID ...........................................................................................9
1.2.1. S lc v c ch hnh thnh cc triterpenoid ......................................9
1.2.2. Phn loi triterpenoid ............................................................................12
1.2.3. c im ca phn ng trong triterpenoid .....................................14
CHNG 2. NGUYN LIU, PHNG TIN, NI DUNG VA PHNG
PHAP NGHIN CU .............................................................................................16
2.1. NGUYN LIU NGHIN CU .................................................................16
2.2. PHNG TIN NGHIN CU ................................................................17
2.2.1. May moc va thit bi nghin cu ............................................................17
2.2.2. Hoa cht, dung mi ................................................................................17
2.3. NI DUNG NGHIN CU .........................................................................17
2.4. PHNG PHAP NGHIN CU ...............................................................17
2.4.1. inh tinh cac nhom cht hu c ...........................................................17
2.4.2. Chit xut phn on cac nhom cht ...................................................18
2.4.3. Phn lp va xac inh cu truc hoa hoc ca cc cht ...........................18
CHNG 3. THC NGHIM VA KT QUA V BN LUN .......................19
3.1. THC NGHIM V KT QUA ................................................................19
3.1.1. inh tinh cac nhom cht trong la u rng ....................................19
3.1.2. Quy trnh chit xut la u rng ......................................................20
3.1.3. inh tnh cn phn on n-hexan/ethylacetat v ethylacetat ...........23

3.1.4. Phn tch cht t cn phn on n-hexan/ethyl acetat bng GC-MS

............................................................................................................................26
3.1.5. Phn lp v nhn dng cht t cn phn on ethylacetat................27
3.2. BN LUN ....................................................................................................35
KT LUN V KIN NGH ................................................................................37
1. KT LUN ...................................................................................................37
2. XUT ......................................................................................................37
TI LIU THAM KHAO ......................................................................................38

DANH MUC CAC K HIU, CH VIT TT

CHCl3
13
C-NMR
d
DC 1
DC 2
DEPT
E
ESI/MS
EtOH
EtOAc
H
HE
1
H-NMR
m/z
MSTB
n-hex
OSC
SKLM
t
TT

Cloroform
Ph cng hng t ht nhn cacbon
Doublet
Dch chit 1
Dch chit 2
Distortionless Enhancement by Polarization Transfer
Cn phn on ethyl acetat
Electrospray ionization/ Mass spectrum
Ethanol
Ethyl acetat
Cn phn on n-hexan
Cn phn on n-hexan/ethyl acetat [5:1]
Ph cng hng t ht nhn proton
T l s khi/ in tch ion
M s tiu bn
n-hexan
Enzym Oxidosqualene cyclases
Sc k lp mng
Triplet
Thuc th

DANH MUC CAC BANG

STT

Tn bng

Trang

Bng 1.1

Cu trc 11 khung cu trc chnh triterpenoid v mt s dn

cht ca chng
Kt qu nh tnh cc nhm cht trong l u rng

12

Kt qu nh tnh cc nhm cht chnh trong l u rng khi

chit xut bng cc dung mi khc nhau
Kt qu nh tnh bng phn ng ha hc ca 2 phn on HE

21

Bng 3.1
Bng 3.2
Bng 3.3

20

24

v E
Bng 3.4

So snh s carbon bc 1, 2, 3 v 4 ca 2 cht glycosid ca

stigmasterol v sitosterol

33

DANH MUC CAC HINH

STT
Hnh 1.1

Hnh 1.2

Tn hnh

Trang

Cu trc aglycon ca cc saponin phn lp t hai loi

Trevesia palmata (Roxb. & Lindl) Vis. v Trevesia sundaica
Miq.
C ch hnh thnh mt s khung cu trc triterpen loi

10

monocylic, bicyclic v tricyclic [26].

Hnh 1.3

C ch hnh thnh mt s khung cu trc triterpen tetracylic

11

Hnh 1.4.

C ch hnh thnh mt s khung cu trc triterpen pentacylic

11

Hnh 2.1

Cy u rng trn thc a v cm hoa

16

Hnh 3.1

Tm tt quy trnh chit xut cc nhm cht trong l u

23

rng
Hnh 3.2

Sc k cn phn on n-Hexan/Ethyl acetat khi trin khai

25

vi h dung mi Toluen/ethyl acetat/acid formic [3 : 1 : 0,1]

Hnh 3.3

Sc k cn phn on ethyl acetat khi trin khai vi h

26

dung mi Toluen/ethyl acetat/methanol/acid formic [8 : 6 :

0,3 : 0,7]
29

Hnh 3.5

Sc k ca TP-ET-02 khai trin vi cc h dung mi I, II,

v III khi hin mu bng TT vanillin/ H2SO4
Ph khi MS-ESI positive ca cht TP-ET-02

Hnh 3.6

Cu trc ca stigmasterol v sitosterol

31

Hnh 3.7

Ph 1H-NMR (500 MHz, DMSO-d6) ca cht TP-ET-02

32

Hnh 3.4

30

(4,95,4 ppm)
Hnh 3.8

Ph 1H-NMR (500 MHz, DMSO-d6) ca cht TP-ET-02

32

(4,24,9 ppm)
Hnh 3.9

Ph 13C-NMR v DEPT ca cht TP-ET-02

34

Hnh 3.10

Cng thc cu to ca -stigmasterol-3-O--Dglucopyranosit

34

Hnh 3.11

Cng thc cu to ca -sitosterol-3-O--D-glucopyranosit

35

T VN
H Nhn sm (Araliaceae) trong h thc vt Vit Nam kh phong ph v a
dng v c im hnh thi cng nh thnh phn ha hc [7]. Hu nh tt c cc
loi trong h Nhn sm u c s dng lm thuc trong Y hc c truyn nhiu
nc trn th gii, c bit l cc nc ng Bc .
Trevesia Vis. l mt chi thuc h Nhn sm, gm khong hn 10 loi, l
nhng cy thuc qu, c gi tr, hin v ang c s dng kh rng ri trong Y
hc dn tc Vit Nam, Trung Quc, Nht Bn, Hn Quc, n v nhiu nc
khc. Cc nh khoa hc xc nh Vit Nam chi Trevesia Vis. gm c 6 loi
phn b rng ri khp c nc, cy thng mc vng rng m, rng xanh hay
dc b sng sui [4].
Trevesia palmata (Roxb. & Lindl.) Vis. , tn thng gi l u rng, Thng
tho gail mt loi thuc chi Trevesia Vis., mc trn cc sn ni co cao
600 2000 m [11], hoc dc theo cc sng, sui, thung lng cc rng phc hi
[2]. u rng c s dng t lu theo kinh nghim dn gian v Y hc c
truyn. Theo ng Y, loi thn co tc dng li tiu, li phu, li sa, dung cha
phu thng, i dt, t thp, lm thuc h nhit, v cng c xem nh mt v thuc
b; l cha gy xng [2]. Tuy nhin Vit Nam, loi cy ny cha c nghin
cu. Trn th gii cho n nay mi ch c mt nghin cu v thnh phn ha hc.
V vy, chng ti thc hin ti Nghin cu thnh phn ha hoc l u
rng (Trevesia palmata (Roxb. & Lindl.) Vis.). thc hin c ti ny, ni
dung nghin cu bao gm:
- nh tnh cc nhm cht hu c trong l u rng;
- Chit xut v chit phn on dch chit l u rng;
- Phn lp v xc nh cu trc hoa hc ca cht phn lp c t l u
rng.

CHNG 1. TNG QUAN

1.1. CHI Trevesia Vis. V LOI Trevesia palmata (Roxb. & Lindl.) Vis.
1.1.1. c im thc vt
1.1.1.1. Vi tri phn loi chi Trevesia Vis.
Theo h thng phn loi ca Takhtajan c ghi trong cun Flowering plants
[22], cy u rng (Trevesia palmata (Roxb. & Lindl.) Vis.) thuc chi Trevesia
Vis., mt chi nm trong h Nhn sm (Araliaceae).V tr phn loi ca chi Trevesia
Vis. trong h thng phn loi thc vt chnh thc co th c tom tt nh sau:
Ngnh Ngc lan Magnoliophyta
Lp Ngc lan Magnoliopsida
Phn lp S Dilleniidae
Lin b Cornanae
B Nhn sm (Hoa tn) Apiales
H Nhn sm (Ng gia b) Araliaceae
Chi Trevesia Vis.
1.1.1.2. c im hnh thi va phn b ca chi Trevesia Vis.
H Nhn sm kh a dng v c im hnh thi. Vit Nam hin nay ghi
nhn c 18 chi thuc h ny, trong o co chi Trevesia Vis. [7].
Chi Trevesia Vis. l nhng cy bi hay cy g nh thng xanh, him khi
phn nhnh; thn c gai ( cy gi, nhng gai ny b tiu bin). L ln, mc so le,
c thy chn vt hay kp chn vt, c cung, mp l kha rng ca; s thy 5 13.
L km dnh vi gc cung l thnh b cha c 2 thy. Cm hoa tn nhiu hoa, hp
li thnh chy hoc chm; tng s tn 1 25, mi tn mang 9 65 hoa. L bc bn
hoc rng sm. Hoa lng tnh. i co mp dng song hay co rng nh. Cnh hoa 6
16, xp van, thng th hp thnh th dng m rng sm. Nh bng s cnh hoa.
Bu di, 6 16 , vi nhy hp thnh ct ngn, mi c 1 non treo. Qu hch
hnh cu trng; ht 1 [2], [11], [16].
Trn th gii, chi Trevesia Vis. co khong hn 10 loi phn b vung ng
Nam A, n , Nepal, Bu-tan, Bng La t v Ty Nam Trung Quc [11], [16].

Di y l khoa nh loi ca chi Trevesia Vis. trong h Araliaceae:

1A. Tin khai hoa xp van.
2B. Bu t hn 20 l non.
3B. L n, x thu chn vt hoc x chn vt.
5B. L khng co m tuyn.
6A. Bu c 5-16 l non
7B. Cy bi hoc g nh, khng c r kh sinh.
8A. Cy c gai .......................................... Trevesia Vis. [7]
1.1.1.3. Hnh thai va phn b ca chi Trevesia Vis. Vit Nam
Chi Trevesia Vis. nc ta co 6 loi, trong o 5 loi c ghi trong cc
sch thc vt Vit nam, o l: Trevesia palmata (Roxb. & Lindl.) Vis., Trevesia
sphaerocarpa Grushv. et Skvorts., Trevesia longipedicellata Grushv. et Skvorts.,
Trevesia cavaleriei (Lv). Grushv. & Skvorts., Trevesia burckii Boerl. [1], [3]. Nm
2007, Phan K Lc v cng s pht hin thm mt loi mi thuc chi Trevesia
Vis. Vit Nam, t tn l Trevesia vietnamensis J. Wen & P.K. Lc [25].
C th phn bit mt s loi thuc chi Trevesia Vis. da trn kha phn loi
sau:
1A. L kp gi ch ngn, phn gc phin l hnh mng.
2A. Trng cnh phn, nhng dnh nhau mt phn to cp i hoc cp ba cnh
hoa...... T. palmata
2B. Trng hoa hp, th dng m, c th rng hon ton .T. burckii
1B. L chia thy hnh ngn, vin thy khng tip xc vi gn chnh ca thy
3A. Hoa v qu trng thnh c cung ngn hoc khng c cung .. T. beccarii
3B. Hoa v qu trng thnh c cung di (>1cm)
4A. Thn cy, cung l v cm hoa ph lng cng dy c, di 1 2 cm
... T. lateospina
4B. Thn cy, cung l v cm hoa ph lp lng ngn hoc mt vi lng cng
5A. Thy l c t l di/rng >3, dng mc; bu 7 8 ... T. valida
5B. Thy l c t l di/rng < 2,5, x thy lng chim nng; bu 8 16

6A. Cung l nhn; mi tn co t hn 15 hoa, bu c 13 16 ... T. arborea

6B. Cung l co vi n nhiu gai; mi tn co hn 20 hoa; bu c 8 12
7A. L c 5 9 thy; trng cnh phn, nhng dnh vi nhau mt phn...
. T. palmata
7B. L c 9 11 thy; cnh hoa hp th dng m, c th rng hon ton
................................... T. sundaica [16]
Tuy nhin hin nay cha co mt kha phn loi no dung phn bit cc loi
trong cng chi Trevesia Vis. Vit Nam.
1.1.1.4. Hnh thi v phn b mt s loi thuc chi Trevesia Vis. Vit Nam
1.1.1.4.1. Trevesia palmata (Roxb. & Lindl.) Vis.
Vit Nam gi l u rng, Nht phin, Thi hoang, Thng tho gai, Thu
du ni [1].
L cy nh cao 7 8 m hoc hn, thn t phn nhnh, cnh c gai, rut xp.
L n, phin l phn thu chn vt, x su nh l thu du, c 5-9 thu nhn c
rng, gn ni hai mt, mp l co rng ca th; cung l di v c gai. L non ph
lng mm, mu nu nht, l gi nhn. Hoa mc thnh tn, t thnh chu nch.
Hoa to khong 1cm, mu trng. Qu di 13-18mm, c kha; ht dt.
Hoa thng 5 6. Qu thng 7 9 [2].
Phn b: u rng mc nhng ch m dc theo cc sng, sui, thung
lng cc rng phc hi [2]. nc ta, loi ny phn b cc tnh Cao Bng, Lng
Sn, Ba V, Qung Tr [3], Sn La, Lo Cai, Tuyn Quang, H Ty, Kon Tum, Gia
Lai, c Lc, Lm ng [1]. Trn th gii, chng c Trung Quc, Bng La t,
Nepal, n , Thi Lan, Lo, Cam Pu Chia [11], [16].
1.1.1.4.2. Trevesia sphaerocarpa Grushv. et Skvorts.
Vit Nam gi l u rng qu trn [2], Nht phin qu trn [1].
L cy nh cao 7 m, thn to 8 cm, khng nhnh hay t nhnh, khng gai. L
non c lng st, c 5 7 thy su; cc thy ny li chia thy nhn, mp co rng;
cung l di n 50 cm, l km 2 cm. Cm hoa nch l, dng chm mang tn trn
trc di 15 cm; mi tn c 20 30 hoa, cung hoa c lng st, di 3 4 cm. Hoa c

11 cnh hoa, 11 12 nh; bu (8-) 10 (-11) . Qu to 2 cm, cung qu di 3 4 cm;

ht to 9 12 mm [2], [3].
Loi ny mc hoang trong rng, thng ni vi cao 500 1200 m,
cc tnh Qung Ninh, Ninh Bnh, Qung Tr, Lm ng, Sn La [1], [2]. C hoa
thng 5, c qu thng 10 [2].
1.1.1.4.3. Trevesia longipedicellata Grushv. & Skvorts.
nc ta gi l cy Nht phin cung di [1].
L cy nh cao 6 m; thn to 10 cm, khng nhnh, c gai. L non c lng st;
phin di n 45 cm; gm 5 7 thuy, co thuy su v co rng nhn; cung l di n
65 cm; l km 2 cm. Chum mang tn hay tn c c; trc hoa 25 cm; tn to 15 cm,
cung hoa di 5 7 cm; hoa 7 9 cnh hoa; 7 9 nh, bu c 7 9 . Qu to 2 cm,
mu en; ht di 7 mm [3].
Cy mc ri rc trong rng, cao 500 m, c Lai Chu, Sn La, Cao
Bng, Lng Sn, Vnh Phc, c Lc, Lm ng [1].
1.1.1.4.4. Trevesia cavaleriei (Lv). Grushv. & Skvorts.
nc ta gi l cy Th d [1].
L cy nh hoc g nh, khng gai. L x chn vt, chia 5 9 thy; thy c
rng, lc non c lng, ri khng lng. Cm hoa l chm mang tn; bu 8 12 .
Qu di 15 18 mm [3].
Loi ny mc Ba V, Ngh Tnh [3], Lo Cai, H Giang, Tuyn Quang, Bc
Cn, Ha Bnh, H Ty, H Ni, Ngh An, Lm ng [1]. Trn th gii, cy cn
thy mc Trung Quc [1].
1.1.1.4.5. Trevesia burckii Boerl.
nc ta gi l cy u rng burck, Thu du ni burck [1].
Cun Cy c Vit Nam, quyn 2 ca Phm Hong H m t rt s lc v
loi ny. L cy bi nh. L chn vt, c 7 thy; thy c cung; cnh v y nhp
vo mt phin chn vt, khng lng, mp co rng tha [3].
Ti liu tng quan chi Trevesia (h Araliaceae) [16] m t rt chi tit loi ny
nh sau: Cy bi cao ti 5 m; t phn nhnh; thn cy c gai mc ri rc, di 0,2

0,7 cm; phn thn non ph lng t mu nu. Cy con co l n, hnh tam gic
trng, cy gi c l chn vt, ging nh hnh cc ngon tay trn mt bn tay. Phin
thy p st gn gia ca tng thy v tch bit vi phn gc l hnh tim; c 7 9
thy hnh mc hay trng ngc, kch thc 15 x 36 cm, nh nhn, mp kha rng
ca. Cung l di 20 50 cm, trn nhn hoc c gai thng mc ri rc. B l 2 cm;
l km 1 2,5 cm; ph lng mu nu . Cm hoa ngn thn hoc gia cc vng
l; mc thnh chm; trc chnh di ti 60 cm, pht sinh 6 12 trc th cp di 23
cm; trn o co mang cc tn n. L bc hnh tam gic, nhn hoc c gai mc ri
rc. Mi tn rng 3 8 cm, mang 30 50 hoa n; cung hoa di 10 35 mm.
Trng c 7 10 cnh hoa, hp thnh th dng m. B nh c 7 10 nh; bao phn
hnh elip, kch thc 2 x 3 mm; ch nh di 2,5 mm. Ht phn kch thc 30 -35 m.
Bu c 7 10 . Qu hnh cu, ng knh 2 cm [16].
Cy mc ri rc trong rng tha, cao di 300 m, mt s tnh thnh nc
ta nh Yn Bi, Ha Bnh, Ngh An, c Lc, Lm ng. Trn th gii, loi ny
cn c Thi Lan, Malaysia, Indonesia (Sumatra, Borneo) [1].
1.1.1.4.6. Trevesia vietnamensis J.Wen & P.K. Lc
Cy cao 3 5 m. Thn c gai hnh nn; thn non ph lng cng di, dy c.
L kp chn vt, gm 4 5 l cht; l km dnh vi gc cung l; cung 20 40 cm,
ph lng cng ri rc. L cht kiu lng chim, vin ngoi hnh trng, kch thc 30
70 x 20 60 cm, mt trn l nhn hoc c lng cng, mt di l co lng t hnh
sao ri rc; cung l cht di 1 4 cm, nh l nhn; gn l ni r c 2 mt l;
mp kha rng ca. Cm hoa pha ngn, gm 3 5 tn trn mt trc chnh di 20
35 cm, ph lng cng di 28 mm, dy c. Mi tn mang 50 110 hoa. L bc
hnh mc. Cung hoa, chi hoa v hoa non ph lng t dy c. Hoa mu trng
hoc trng hi xanh. i hoa hnh tam gic, di 0,9 1,1 mm, rng 0,6 0,7 mm.
Trng 4 5 cnh, kch thc 5,0 5,5 x 2,5 2,8 mm. B nh 4 5, bao phn thun
di 2,0 2, 2 mm. Bu c 4 5 . Qu hnh cu trng ngc, di 8 9 mm, rng
7 8 mm.
C hoa cui thng 9. Qu thng 3.

Loi ny a nhng ni mt m, m t, nhiu mun, trong cc thung lng v

ni vi co cao 500 850 m. Hin thy mc tnh Thanh Hoa, Cc Phng
(Ninh Bnh), Mc Chu (Sn La) [25].
1.1.2. Thanh phn hoa hoc ca chi Trevesia Vis. va cy u rng
Cho n nay nhng nghin cu v thnh phn hoa hc ca cc loi trong chi
Trevesia Vis., k c trong nc v nc ngoi vn cn rt hn ch.
Chng ti mi ch tm c hai nghin cu nc ngoi v thnh phn saponin
ca hai loi thuc chi Trevesia Vis., o l loi T. palmata (Roxb. & Lindl.) Vis. v
loi T. sundaica Miq.
Nm 1997, t phn on n- BuOH ca dch chit MeOH t hoa v l loi T.
sundaica Miq. (thu hi Italia), Nunziatina De Tommasi v cng s (Italia) phn
lp c 10 saponin triterpenoid [13]. Nm 1999, cng t phn on n- BuOH ca
dch chit EtOH t l loi T. palmata (Roxb. & Lindl.) Vis. (thu hi Italia), nhm
nghin cu ny phn lp c 8 saponin triterpenoid [12]. Phn ln cc saponin
ny l cc bisdesmosid, ch c 3 saponin monodesmosid, nhng tt c u c phn
aglycon l acid oleanolic hoc dn cht ca acid ny.

Hnh 1.1. Cu trc aglycon ca cc saponin phn lp t hai loi

Trevesia palmata (Roxb. & Lindl) Vis. v Trevesia sundaica Miq.
v tr R2 l nhm methyl (CH3) hoc nhm th alcol (CH2OH); R3 l hydro
(H) hoc nhm th hydroxy (OH).

Trong loi T. palmata (Roxb. & Lindl.) Vis., mch ng ca cc saponin

gm 2 3 n v monosaccarid lin kt vi C3 (lin kt glycosid) v i vi cc
bisdesmosid, c 3 4 n v monosaccarid lin kt vi C28 (lin kt ester) ca phn
genin. Trong khi o loi T. sundaica Miq., mch ng lin kt vi C3 v C28
ln lt l 4 5 v 1 2 n v monosaccarid. Cc n v ng thuc loi
pyranose: -D-glucose v -L-rhamnose c 2 loi; -D-galactose v -D- xylose
loi T. sundaica Miq.; -L-arabinose v -D-quinovose loi T. palmata (Roxb. &
Lindl.) Vis.
Tuy nhin mt nghin cu khc Thi Lan s dng cc phn ng nh tnh
cc nhm cht ha hc c trng, cho thy trong cy Trevesia palmata (Roxb. &
Lindl.) Vis. (thu hi Thi Lan) co triterpen t do, lacton glycosid; khng co
saponin, flavonoid, tanin, alcaloid [17].
1.1.3. Tac dung va cng dung ca cy u rng
Hin nay trn th gii co rt t nghin cu v tc dng dc ly ca u rng
(Trevesia palmata (Roxb. & Lindl.) Vis.).
Nm 1999, Nunziatina De Tommasi v cng s (Y) chng minh dch chit
saponin th t l u rng v mt s saponin phn lp c u th hin tc dng
chng tng sinh t bo trn cc dng t bo J774, HEK-293c, WEHI-164d [12].
Mt nghin cu Thi Lan cho thy dch chit ethanol phn trn mt t
cy u rng co tc dng chng oxy hoa rt yu trn in vitro [17].
nc ta, u rng c s dng t lu theo kinh nghim dn gian v Y
hc c truyn. Theo ng Y, loi thn co tc dng li tiu, li phu, li sa, dung
cha phu thng, i dt, t thp, lm thuc h nhit, v cng c xem nh mt v
thuc b (20 30 g loi thn sc ring hoc phi hp vi cy Mua ); l c dung
nu nc xng cha t lit bi ngi v gi p cha gy xng ( Trung Quc
ngi ta dung l cha ng tn thng hay dao chm thng tch) [2].

1.2. TRITERPENOID
1.2.1. S lc v c ch hnh thanh cac triterpenoid
Triterpenoid l mt nhm hp cht t nhin rng ln, c phn loi thnh
hn 100 khung cu trc khc nhau. S khc nhau v cu trc bt ngun t con
ng sinh tng hp vi s tham gia ca h thng enzym chung c t tn l
terpenoid synthase (ch yu l enzym oxidosqualen cyclases OSC). Cc enzym
ny gip cho s hnh thnh cc carbocation t cc tin cht mch h, bng cch
cng thm tc nhn i in t vo ni i hoc vng epoxy (oxiran), t o to thnh
cc vng hay dn ti s sp xp li b khung cu trc. Cui cng cc cation trung
gian c trung ha in tch bng cch loi proton hoc phn ng vi nc
hnh thnh sn phm. Hai tin cht ph bin nht cho qu trnh tng hp triterpen l
squalen v oxidosqualen [15].
Cc triterpen to thnh c mi quan h mt thit vi nhau. Ngoi c tng
hp trc tip t cc tin cht, chng cn l cc cht trung gian chuyn ha ca nhau.
Chng hn cc triterpen pentacyclic (5 vng) c th b thoi vng di tc dng ca
enzym OSC hnh thnh cc triterpen tetracyclic (4 vng); sau o triterpen
tetracyclic c th b thoi vng hnh thnh cc triterpen tricyclic (3 vng), sn
phm sau khi thoi vng gi l cc seco- [15].
S a dng v cu trc cn c th hin ch cc triterpen c th b thoi
phn mt vi nhom methyl hnh thnh cc phn t c s khi nh hn. Chng
hn triterpen nhm oleanan (30C) b kh mt nhm methyl v tr C30 hnh thnh
triterpen noroleanan c 29 nguyn t carbon.
Hnh 1.2, 1.3 v 1.4 trnh by qu trnh hnh thnh mt s khung cu trc
triterpen loi monocylic, bicyclic, tricyclic, tetracyclic v pentacyclic.

10

Hnh 1.2. C ch hnh thnh mt s khung cu trc triterpen loi monocylic,

bicyclic v tricyclic [26].

11

Hnh 1.3. C ch hnh thnh mt s khung cu trc triterpen tetracylic [26]

Hnh 1.4. C ch hnh thnh mt s khung cu trc triterpen pentacylic [26].

12

1.2.2. Phn loi triterpenoid

Phn loi triterpenoid ch yu da vo phn aglycon.
Hu ht cc triterpen trong t nhin c cu trc 5 vng (pentacyclic) hoc 4
vng (tetracyclic) [26]. Hai loi cu trc ny c xp vo nhm cht saponin nh
tnh cht to bt. Theo cch phn loi mi nht hin nay (2010) [14], triterpenoid
c chia thnh 11 nhm chnh l oleanan, ursan, hopan, lupan, taraxastan,
dammaran, lanostan, tirucallan, cucurbitan, cycloartan v holostan. Trong o nhom
oleanan l nhm ln nht.
Thc t cc triterpen mch h, 1 vng, 2 vng, 3 vng v 6 vng cng c
phn lp t thc vt [26]. V d gn y cc nh khoa hc phn lp c cc
triterpenoid c cu trc 3 vng (tricyclic) nh cc cht kadcotriones A C c trong
thn loi Kadsura coccinea (Lem.) A. C. Smith [19] v cht schiglautone A trong
thn loi Schisandra glaucescens Diels., h Ng v t (Schisandraceae) [21]; phn
lp cht achilleol A c cu trc 1 vng t loi Camellia sasanqua [9]; cht
myrrhanol A c cu trc 2 vng t loi Balsamodendron mukul [20]; 6 triterpenoid
c cu trc 6 vng (hexacyclic) t loi Euscaphis japonica [10].
Bng 1.1 trnh by cu trc 11 khung cu trc chnh ca nhm triterpenoid v
mt s dn cht ca chng.
Bng 1.1. Cu trc 11 khung cu trc chnh triterpenoid v mt s dn
cht ca chng [24]

Olean

17, 22-seco olean

13

Olean bin i

30-nor olean

Ursan

18, 19-seco ursan

Hopan

Bis-nor hopan

Lupan

3,4-seco lupan

14

Dammaran

15,16-seco dammaran

Taraxastan

Cycloartan

Tirucallan

Cucurbitan

Lanostan

Holostan

1.2.3. c im ca phn ng trong triterpenoid

Cu trc phn ng in hnh ca cc triterpenoid pentacyclic v tetracyclic
l cc chui ng oligo 2 5 n v monosaccarid, thng lin kt vi phn genin

15

v tr C3 theo lin kt glycosid hoc/v C28 theo lin kt ester. Mch ng lin
kt vi phn genin v tr C6, C7, C20, C21, C22, C24 hoc C26 theo lin kt
glycosid gp cc triterpenoid c cu trc 4 vng (tetracyclic).
Cc kt qu nghin cu co cho thy cc cu trc triterpenoid tricyclic,
bicyclic, monocyclic u khng gn thm cc mch ng.
Cc monosaccarid ph bin nht, hay gp trong triterpenoid l cc ng
thng thng nh glucose, acid glucoronid, rhamnose, xylose, arabinose, galactose.
Him gp hn l cc ng quinovose, fucose, arabifuranose, ribose, apiose.
Hu ht cc triterpenoid l nhng monodesmosidic (c 1 mch ng) hoc
bidesmosidic (c 2 mch ng). Ngoi l mt s loi thc vt c triterpenoid
tridesmosidic (c 3 mch ng), chng hn nh loi Chenopodium quinoa Willd.,
loi Astragalus armatus.

16

CHNG 2. NGUYN LIU, PHNG TIN, NI DUNG

VA PHNG PHAP NGHIN CU
2.1. NGUYN LIU NGHIN CU
Mu nghin cu l l cy u rng thu hi huyn V Xuyn, tnh H
Giang vo thng 1 nm 2014. Dc liu sau khi thu hi em ra sch, ct nh phi
ni thong mt, ri sy kh 55 60oC trong t sy co qut thng gio. Sau khi
sy kh, dc liu c nghin thnh bt th v bo qun trong ti nilon kn, ni
thong mt, kh ro.
Mu nghin cu co hoa c lm tiu bn, lu ti Phng tiu bn B mn
Thc vt, trng i hc Dc H Ni, MSTB: HNIP/18064/14. TS. Nguyn Quc
Huy - b mn Thc vt, Trng i hc Dc H Ni, gim nh tn khoa hc
mu nghin cu l Trevesia palmata (Roxb & Lindl) Vis., thuc h Nhn sm
(Araliaceae).

Hnh 2.1. Cy u rng trn thc ia v cum hoa

17

2.2. PHNG TIN NGHIN CU

2.2.1. May moc va thit bi nghin cu
Sy dc liu bng t sy SHELLAB
My chit siu m Extractor - Sony SM30 CEP, tn s 60 MHz
T sy BINDER nhit 105 110oC
My ct quay Buchi ROTAVAPOR R 200
Cn k thut Sartorius
My xc nh m SARTORIUS
Bn mng trng sn silica gel GF254 ca hng MERCK (c)
Cc dng c th nghim thng thng (bnh gn, cc co m, pipet, ng ong)
My o khi ph LC/MS/MS Xevo TQMS ti Vin ha hc
My o ph cng hng t ht nhn 1H-NMR v

13

C-NMR BRUKER

AVANCE AM500 FT-NMR ti Vin ha hc.

2.2.2. Hoa cht, dung mi
Cc hoa cht do phng Gio ti i hc Dc H Ni cung cp.
Cc dung mi t chun phn tch, th nghim.
2.3. NI DUNG NGHIN CU
-

nh tnh cc nhom hp cht hu c trong l u rng

Chit xut v chit phn on dch chit l u rng

Phn lp v xc nh cu trc hoa hc ca cc cht phn lp c t dch

chit l u rng.
2.4. PHNG PHAP NGHIN CU
2.4.1. inh tinh cac nhom cht hu c
Chit xut v nh tnh cc nhom cht hu c trong l u rng bng cc
phn ng hoa hc c trng theo cc ti liu Phng phap nghin cu hoa hoc cy
thuc [6] v Dc liu hoc tp 1 [5].
nh tnh cc phn on bng sc k lp mng s dng bn mng trng sn
silicagel GF254. Quan st bn mng di nh sng thng, UV254, UV366 v sau o
hin mu bng thuc th vanillin/H2SO4 (h nong).

18

2.4.2. Chit xut phn on cac nhom cht

-

Chit xut cc nhom cht trong l u rng bng dung mi ethanol 70%,

sau o ct thu hi dung mi di p sut gim.

-

Chit phn on: dch chit ton phn ln lt c chit vi cc h dung

mi hu c co phn cc tng dn: n-hexan (H), n-hexan/ethylacetat [5:1] (HE)

v ethylacetat (E).
2.4.3. Phn lp va xac inh cu truc hoa hoc ca cac cht
-

Phn tch phn on HE bng phng php GC-MS.

Phn lp cc hp cht trong phn on E bng sc k ct vi cht hp ph l

silicagel, c ht 0,040 0,063 mm.

13

Theo doi cc phn on chy ct bng sc k lp mng.

Nhn dng cc cht phn lp c da trn d liu cc ph MS, 1H-NMR,

C-NMR, DEPT; so snh vi thng tin trong cc th vin ph.

19

CHNG 3. THC NGHIM VA KT QUA VA BAN LUN

3.1. THC NGHIM VA KT QUA
3.1.1. inh tinh cac nhom cht trong la u rng
Tin hnh nh tnh xc nh cc nhm cht hu c c mt trong l u
rng da vo cc hin tng vt l v cc phn ng ha hc c trng sau:
nh tnh saponin: quan st hin tng to bt v phn bit 2 loi saponin
da trn tnh cht to bt trong mi trng acid v kim.
nh tnh flavonoid: s dng phn ng cyanidin; phn ng vi FeCl3 5%;
vi dung dch kim long NaOH 10% v hi amoniac.
nh tnh alcaloid: s dng cc thuc th Mayer, Bouchardat v
Dragendorff.
nh tnh anthranoid dang t do v dang ton phn: s dng phn ng
vi dung dch kim long (phn ng Borntraeger).
nh tnh coumarin: s dng phn ng m - ong vng lacton; phn ng
vi thuc th diazo; quan st hin tng hunh quang v vi thng hoa.
nh tnh glycosid tim: s dng phn ng Liebermann (khung steroid);
phn ng Legal (vng lacton) v phn ng Keller Kiliani (ng 2,6 - desoxy).
nh tnh tanin: s dng phn ng vi dung dch FeCl3 5%; vi dung dch
ch acetat 10% v vi dung dch gelatin 1%.
nh tnh polysaccarid: s dng phn ng vi TT Lugol.
nh tnh ng kh: s dng phn ng vi TT Fehling A + Fehling B.
nh tnh acid hu c: s dng phn ng vi bt Na2CO3.
nh tnh cht bo: bng cch quan st hin tng vt m trn giy lc khi
nh dch chit ln ming giy lc.
nh tnh caroten: s dng phn ng vi H2SO4 c.
nh tnh sterol: s dng phn ng Liebermann- Burchard.
Kt qu nh tnh cc nhm cht trong l u rng thu hi H Giang c
trnh by bng 3.1.

20

Bng 3.1. Kt qu inh tnh cc nhm cht trong la u rng

TT

Nhm cht

Kt qu inh tnh

Kt lun

Triterpenoid (+++)

C saponin triterpenoid

Saponin

Flavonoid

Khng c

Alcaloid

Khng c

Coumarin

Khng c

Glycosid tim

Khng c

Anthranoid

Khng c

Tanin

Khng c

Polysaccarid

++

ng kh

+++

10

Acid hu c

++

11

Cht bo

++

12

Caroten

Khng c

13

Sterol

+++

Ghi ch: (-) Phn ng m tnh

(+) Phn ng dng tnh

(++) Phn ng dng tnh ro

(+++) Phn ng dng tnh rt r

Nhn xt: Trong l cy u rng (Trevesia palmata Roxb ex Lind. Vis) c cc

nhm cht: saponin triterpenoid, acid hu c, ng kh, sterol, polysaccarid v
cht bo. Trong o saponin triterpenoid l nhm cht chnh.
3.1.2. Quy trnh chit xut la u rng
3.1.2.1. Kho st dung mi chit xut
Da trn kt qu nh tnh cc nhm cht hu c chnh, chng ti nhn thy l
cy u rng c 3 nhm cht co tnh tan khc nhau, o l nhom saponin
triterpenoid (+ sterol); nhm th hai l ng kh + polysaccarid + acid hu c;
nhm th ba l cht bo. Trong 3 nhm ny, hai nhom u tan tt trong dung mi
nc v cn nc, nhm th 3 tan trong dung mi hu c t phn cc. Do vy,
chng ti kho st chit bng 4 h dung mi sau: nc; EtOH 30%, 70%, v 96%.

21

Quy trnh chit xut vi mi h dung mi c thc hin nh sau: Cn chnh

xc khong 30 g bt l u rng ( m 9,41%), thm m dc liu bng dung
mi chit. Sau o cho nguyn liu chit vo bnh cu, sinh hn v thm dung mi
chit ngp dc liu khong 1,5 th tch (khong 200 mL), chit hi lu trong 5 h.
Rt dch chit, lc. Dch chit thu c tin hnh nh tnh cc nhm cht chnh:
triterpenoid (hin tng to bt), ng kh, polysaccarid, acid hu c, cht bo v
chlorophyll. V nguyn liu nghin cu l l nn chlorophyll chim lng ln v
nh hng rt nhiu n qu trnh chit xut v phn lp.
Kt qu nh tnh cc nhm cht chnh ca 4 dch chit nc, EtOH 30%,
EtOH 70% v EtOH 96% c trnh by bng 3.2.
Bng 3.2. Kt qu inh tnh cc nhm cht chinh trong la u rng
khi chit xut bng cc dung mi khc nhau.
TT

Nhm cht

Dung mi chit xut

Nc

EtOH 30%

EtOH 70%

EtOH 96%

1.

Triterpenoid

+++

+++

+++

2.

ng kh

+++

+++

3.

Polysaccarid

++

++

4.

Acid hu c

++

++

5.

Cht bo

++

6.

Chlorophyll

++

+++

Nhn xt: Khi chit xut bng dung mi EtOH 96% th lng cht chnh thu
c t v ln nhiu tp tan trong dung mi hu c km phn cc l chlorophyll v
cht bo. Khi chit bng dung mi nc v cn 30% th thu c lng cht chnh
ln nhng dch chit ln nhiu tp tan tt trong nc nh ng kh v acid hu
c. Hn na, dch chit thu c kh ct thu hi dung mi. S dng dung mi
EtOH 70% c nhiu u im l t tp hn, thu c nhm cht chnh v dch chit
d ct thu hi dung mi.

22

3.1.2.2. Chit xut v chit phn on

Da vo kt qu kho st dung mi chit xut trn, chng ti la chn EtOH
70% chit xut dch chit ton phn.
Quy trnh chit xut c thc hin nh sau: Cn 1,0 kg bt l ( m 9,41%)
cho vo bnh chit siu m 10 L, thm 1,5 L ethanol 70% thm m dc liu,
trn u. Sau o thm 4 L dung mi dung mi chit ngp hon ton nguyn liu
(khong 1,3 ln th tch dc liu). Tin hnh chit siu m vi thi gian chit mi
ln l 90 pht, nhit 60oC, tn s siu m 60 MHz. Rt dch chit ln th nht,
tip tc cho thm dung mi chit ln 2 v ln 3 vi iu kin chit nh ln th
nht. Gp dch chit, lc thu c 11 L dch chit cn (k hiu: DC1). Ct thu hi
dung mi dch chit DC1 di p sut gim n khi cn 3 L thu c dch chit 2
(k hiu: DC2). Tin hnh chit kit DC2 ln lt vi cc h dung mi co phn
cc tng dn: n-hexan, n-hexan/ethyl acetat [5:1] v ethyl acetat (mi dung mi tin
hnh chit 3 ln) thu c 3 phn on dch chit. Cc phn on dch chit c
ct thu hi dung mi di p sut gim thu c cn phn on n-hexan (k hiu H;
1,1 g), cn phn on n-hexan/ethylacetat [5:1] (k hiu HE; 2,5 g), cn phn on
ethylacetat (k hiu E; 6,1 g). Quy trnh chit xut v chit phn on l u rng
c trnh by hnh 3.1.

23

Hnh 3.1. Tm tt quy trnh chit xut cc nhm cht trong la u rng
Nhn xt: cn phn on dch chit n-hexan thu c c khi lng t nht, th
cht dnh, mu xanh en. Khi th nh tnh phn on ny bng sc k lp mng,
trn sc ky ch c vt mu xanh v (UV 366 nm). Do vy c th s b kt
lun phn on ny ch yu l chlorophyll, cht bo nn trong kha lun ny chng
ti khng tin hnh nghin cu tip. Cn phn on n-hexan/ethylacetat v
ethylacetat c khi lng l 2,5 g v 6,1 g tng ng c s dng nghin cu
su hn.
3.1.3. inh tinh cn phn on n-hexan/ethylacetat v ethylacetat
3.1.3.1. nh tnh bng phn ng ha hoc
Kt qu nh tnh phn on n-hexan/ethylacetat v ethylacetat bng phn
ng ha hc c trnh by bng 3.3.

24

Bng 3.3. Kt qu inh tnh bng phn ng ha hoc ca 2 phn on HE v E

Phn on
STT

Phn ng inh tnh

Nhm cht

HE

Saponin triterpenoid

Hin tng to bt

Sterol

Liebermann

+++

Cht bo

Vt m trn giy lc

++

ng kh

TT Fehling A+B

Acid hu c

Na2CO3

Polysaccarid

TT Lugol

Nhn xt: Bng phn ng ha hc c trng, chng ti nhn thy cc nhm cht
tan tt trong nc nh ng kh, acid hu c v polysaccarid u khng c mt
2 phn on n-hexan/ethyl acetat v ethyl acetat. C 2 phn on dch chit u
khng c hin tng to bt. Ch c nhm sterol c mt c 2 phn on, trong o
phn on ethyl acetat cho phn ng dng tnh ro nht.
3.1.3.2. nh tnh bng sc k lp mng
nh tnh cn phn oan n-hexan/ethyl acetat
Dch chm sc k: Ha tan cn HE trong MeOH lm dch chm sc k.
Cht hp ph: bn mng silicagel GF254 (Merck) trng sn hot ha
110oC trong 1 gi.
Cc h dung mi khai trin:
H I: n-hexan/ethyl acetat

[5 : 1]

H II: n-hexan/ethyl acetat

[1 : 1]

H III: Toluen/ethyl acetat/acid formic

[5 : 1 : 0,1]

H IV: Toluen/ethyl acetat/acid formic

[3 : 1 : 0,1]

Hin mu: quan st di nh sng UV 254 v 366 nm; sau o phun TT

vanillin/H2SO4 ri sy 110oC trong 10 pht.
Kt qu cho thy h dung mi IV cho sc ky c cc vt tch r rng v
nhiu vt nht. Hnh 3.2 trnh by sc ky ca cn phn on HE khi trin khai

25

vi h dung mi IV v quan st cc iu kin khc nhau (nh sng thng, UV

254, 365 nm v TT vanillin/ H2SO4).

AS thng

UV254

UV365

TT Vanilin/H2SO4

Hnh 3.2. Sc ki cn phn on n-Hexan/Ethylacetat khi trin khai vi

h dung mi Toluen/ethyl acetat/acid formic [3 : 1 : 0,1]
nh tnh cn phn oan ethyl acetat
Tng t nh trn, dch chit cn phn on ethylacetat c nh tnh bng
SKLM vi cc h dung mi khai trin khc nhau nh sau:
H I: Diclomethan/ethyl acetat

[1 : 4]

H II: Cloroform/ethyl acetat/acid formic

[8 : 1 : 0,5]

H III: Cloroform/methanol

[9 : 1]

H IV: Ethyl acetat/methanol/acid formic

[10 : 0,2 : 0,1]

H V: Toluen/ethyl acetat/methanol/acid formic

[8 : 6 : 0,3 : 0,7]

Hin mu: quan st di nh sng UV 254 v 366 nm; sau o phun TT

vanillin/H2SO4 ri sy 110oC trong 10 pht.

26

Kt qu cho thy h dung mi V cho sc ky c cc vt tch r rng v

nhiu vt nht. Hnh 3.3 trnh by sc ky ca cn phn on E khi trin khai vi
h dung mi V v quan st cc iu kin khc nhau.

AS thng

UV254

UV365

TT vanilin/H2SO4

Hnh 3.3. Sc ki cn phn on ethyl acetat khi trin khai vi h dung mi

Toluen/ethyl acetat/methanol/acid formic [8 : 6 : 0,3 : 0,7]
3.1.4. Phn tich cht t cn phn on n-hexan/ethyl acetat bng GC-MS
Theo mt s nghin cu nc ngoi, c th phn tch thnh phn sterol trong
cc phn on dch chit km phn cc bng phng php sc k kh (GC) hoc sc
k kh kt hp khi ph (GC MS) [8] [23]. Do vy, chng ti th phn tch phn
on co phn cc thp l n-hexan/ethyl acetat [5:1] bng phng php GC - MS.
trnh lm tc ct sc k kh, chng ti tin hnh tinh ch cn phn on
HE qua sc k ct m vi cht nhi ct l silicagel, dng dung mi n-hexan (PA)
ra gii. Sau o dch ra gii thu c, c ct thu hi dung mi ti cn kh ri
ha tan vo dung mi n-hexan phn tch.

27

iu kin phn tch:

-

H thng GC: Agilent Technologies 7890A

H thng MS: Agilent Technologies 5975C

Ct sc k: HP-5MS. Chiu di ct: 30 m, ng knh ct: 0,25 mm

Chng trnh khai trin:

-

Mu phn tch: TPHE

Tim mu t ng

Th tch tim mu: 1 microlit, khng chia dng

S dng kh mang Heli, tc kh mang: 1 ml/pht

Chng trnh nhit :

Thi gian (pht)

Nhit (oC)

0-1

50

1-41

50-250

41-61

250

Ct
Nhit hoa hi
-

290

Detector: MS
Ph khi c so snh vi cc th vin Wiley, Flavor v Nist phn tch kt

qu. Kt qu cho thy c 62 phi t c pht hin trong mu phn tch. Trong o
a s l cc dn cht alkan nh octan, cyclohexan, dodecan, nonan, octadecan,
hexadecane, eicosan
3.1.5. Phn lp va nhn dng cht t cn phn on ethyl acetat
3.1.5.1. Phn lp
Chun b mu: S dng cn phn on ethylacetat tin hnh phn lp cc
cht. Cn E c ha tan trong mt lng ti thiu ethyl acetat ri trn vi mt t
silicagel, sau o ct thu hi dung mi p sut gim, thu c mt hn hp bt
kh, ti, trn chy tt, mu vng xanh.
Chun b ct: Ct thy tinh c kha v np y kn, ng knh 4cm, chiu
di 60cm. Ra sch ct, sy kh.

28

Cht nhi ct: Ly khong 60g silicagel kch thc 0,04 0,063 mm dng cho
sc k ct. Hot ha 110oC trong 1 gi, sau o ngui trong bnh ht m.
Nhi ct bng phng php nhi ht t: Dng dung mi n-hexan to hn
dch ng nht vi cht hp ph silicagel. Np vo ct. Dng kh nn lp
silicagel c nn cht v n nh.
Np mu kh: a t t bt mu chun b ln ct. Sau o tin hnh ra gii
ct vi h dung mi n-hexan/ethyl acetat gradient nng , t l t [5 : 1], [3 : 1], [1
: 1], cui cng l [1 : 4].
Dung dch ra gii c hng tng 20 mL vo ng nghim v kim tra bng
sc k lp mng. Cc ng nghim c sc k tng t c gp chung thnh mt
phn on. Kt qu thu c 3 phn on ng ch y, k hiu l E1, E2 v E3.
Phn on E1 t ng s 22 ti ng s 31, thy xut hin tinh th hnh li
lim. em lc, ra, thu tinh th khng mu, k hiu TP-E1-1. Cht ny t tan trong
ethyl acetat, tan c trong methanol.
Phn on E2 t ng s 94 ti ng 97, thy c ta trng. em lc v ra ta
nhiu ln bng n-hexan thu c bt v nh hnh mu trng, k hiu TP-E2-1.
Phn on E3 t ng s 247 ti ng s 292, kim tra bng sc k lp mng vi
h dung mi khai trin ethyl acetat/methanol/acid formic [10:0,2:0,1], pht hin
bng TT vanillin/H2SO4 thy xut hin 2 vt mu tm, trong o co 1 vt m v 1
vt nht hn. Ct thu hi dung mi cn khong 5 mL, kt tinh lnh qua m, thu
c cht v nh hnh mu trng. Lc v ra nhiu ln vi dung mi n-hexan/ethyl
acetat [1 : 1] lnh, thu c bt v nh hnh mu trng, k hiu TP-ET-02.
Do lng cht TP-E1-0 v TP-E2-1 t v thi gian cha cho php nn trong
nghin cu ny chng ti khng x l tip cc cht ny. Cht rn TP-ET-02 c
kim tra tinh khit v o ph xc nh cu trc.
3.1.5.2. Kim tra tinh khit ca cht TP-ET-02
Kim tra tinh khit ca cht TP-ET-02 bng sc k lp mng, khai trin bn
mng vi 3 h dung mi sau:
H I: Toluen/ethyl acetat/methanol/acid formic

[ 8 : 6 : 0,3 : 0,7]

29

H II: Cloroform/ethyl acetat/acid formic

[4 : 1 : 0,5]

H III: Cloroform/methanol

[4 : 1]

Kt qu cho thy vi cc h dung mi khai trin khc nhau, cht TP-ET-02

khng quan st c nh sng thng, cng nh nh sng t ngoi 254 nm, v
u ch cho 1 vt mu tm trn sc k sau khi hin mu bng thuc th
vanillin/H2SO4. Cht TP-ET-02 c Rf ln lt 0,17; 0,31; v 0,64 tng ng vi 3
h dung mi khai trin. Hnh 3.4 l sc ky ca cht TP-ET-02.

Hnh 3.4. Sc ki ca TP-ET-02 khai trin vi cc h dung mi I, II,

v III khi hin mu bng TT vanillin/ H2SO4
Do vy c th s b kt lun rng cht TP-ET-02 l tinh khit.
3.1.5.3. Nhn dng cht TP-ET-02
Tnh cht l ha: th v nh hnh mu trng. t tan trong EtOAc v CHCl3,
tan trong MeOH.
Ph khi (MS-ESI): ph negative cht TP-ET-02 cho pic phn t [M-H2H2O]+ = 609 m/z v ph positive cho pic phn t [M+Na]+ = 597 m/z, vy [M]+ =
574 m/z. S d chng ti phi o ph khi 2 ln vi 2 cch o khc nhau co c
thng tin pic phn t. ln o th nht, ph negative cho pic phn t c s khi l

30

609 m/z, thng thng s khi ny l s khi ca pic [M-H]+, nhng khi so s khi
ny vi ph proton 1H-NMR ca cht TP-ET-02 chng ti khng thy ph hp nn
xut o ph khi positive. Kt qu ph khi positive ph hp vi khung cu
trc chng ti d on hn (co 35 tn hiu carbon trn ph 13C-NMR) , c bit vic
xut hin pic mnh c s khi m/z 413 tng ng vi [M-ng+H]+. Do vy cht
TP-ET-02 c cng thc phn t l C35H58O6 hay C29H47 O C6H11O5, trong o
phn ng c d on l ng glucose v phn aglycon c d on l
stigmasterol hoc ng phn ca stigmasterol.

Hnh 3.5. Ph khi MS-ESI positive ca cht TP-ET-02.

Thc vy ph 1H-NMR v 13C-NMR cht TP-ET-02 c o trong dung mi
DMSO-d6, tn s 500 MHz cho tn hiu c trng ca khung cu trc
stigmasterol hoc ng phn ca stigmasterol v 1 phn t ng glucose. Nh vy
phn aglycon c th l stigmasterol, hoc -sitosterol hoc spinasterol.

31

d on phn aglycon c cu trc sterol chng ti da vo 3 vng tn

hiu trn ph 1H-NMR nh sau: tn hiu ca cc nhm methyl; tn hiu ca proton
olefin trong vng; tn hiu ca proton olefin ngoi vng (phn bit cu trc
stigmasterol vi -sitosterol).
Trn ph 1H-NMR ca cht TP-ET-02 xut hin tn hiu ca 6 nhm metyl
trong o co 2 tn hiu singlet ti dch chuyn ha hc l 0,67 v 0,96 ppm (tng
ng vi H-18 v H-19), 3 tn hiu doublet ti 0,78 (H-27), 0,83 (H-26) (c cng
hng s tng tc J = 5,0 Hz l tng tc germinal ca 2 nhm metyl gn vo nhm
metin (CH3-CH)) v 1,00 (H-21) ppm (J = 6,5 Hz) v 1 tn hiu triplet ti 0,78
ppm (H29) l tng tc ca nhm metyl gn vo nhm metylen (CH3-CH2). Vi tn
hiu 6 nhm metyl ny cho chng ti nhn dng s b ban u cht TP-ET-02 c
cu trc sterol. phn bit vi triterpenoid cu trc 5 vng, v d nh nhom
oleanan cc tn hiu ca nhm metyl l singlet v c 7 nhm metyl.
Hai cht stigmasterol v -sitosterol c cng thc nh hnh di y, thc t
trn ph proton ch khc nhau tn hiu ca proton olefin mch nhnh (H-22 v
H-23), cn li cc tn hiu ca 6 nhm metyl v proton H-6 l ging nhau.

Stigmasterol

-Sitosterol

Hnh 3.6. Cu trc ca stigmasterol v sitosterol

Trn ph 1H-NMR vng t 5,0 5,4 ppm, c nhm tn hiu ca 3 proton
olefin ti 5,32 ppm (H-6) v 5,15 ppm v 5,05 ppm l 2 proton olefin v tr trans
c hng s tng tc J = 15 Hz. y l tn hiu ca 3 proton olefin ca phn
stigmasterol.

32

Hnh 3.7. Ph 1H-NMR (500 MHz, DMSO-d6) ca cht TP-ET-02

(4,95,4 ppm)
Tip theo, vung trng t 4 4,9 ppm cho tn hiu ca proton anomer ti
4,2 (J=7,5 Hz) cng vi vng tn hiu proton 4,39 v 4,84 ppm cho thy s c
mt ca 1 phn t ng glucose trong phn t hp cht (hnh 3.8).

Hnh 3.8. Ph 1H-NMR (500 MHz, DMSO-d6) ca cht TP-ET-02

(4,24,9 ppm)
Nh vy, ban u da trn ph 1H-NMR v ph khi, chng ti nhn dng
cht TP-ET-02 l mt sterol glycosid, c phn aglycon l stigmasterol v phn
ng l glucose. Tuy nhin s proton tng ng vi mi tn hiu pic cho thy s
proton trong phn t ln hn so vi cng thc phn t d oan c t ph khi.

33

Tip tc, chng ti phn tch d liu ph 13C-NMR v ph DEPT 90 v 135.

Ph carbon cho 35 tn hiu, kt hp vi ph DEPT 135, xc nh c c 12 nhm
CH2, trong khi o nu cht TP-ET-02 l stigmasterol vi 1 phn t ng th ch c
10 nhm CH2.
Bng 3.4. So snh s carbon bc 1, 2, 3 v 4 ca 2 cht glycosid ca
stigmasterol v sitosterol.
S nhm

Stigmasterol + glucose

-Sitosterol + glucose

CH3

CH2

10 (trong o 1 CH2 ca ng

12 (trong o 1 CH2 ca ng

glucose)

glucose)

16 (trong o 5 CH ca ng

14 (trong o 5 CH ca ng

glucose)

glucose)

C bc 4

Tng s

35 carbon

35 carbon

CH

Chng ti tm tn hiu carbon gn vo 2 proton olefin (H-22 v H-23) ca

stigmasterol th thy c 2 tn hiu rt yu ti 138 ppm v 129 ppm, tn hiu ny nh
hn hn so vi cc tn hiu carbon cn li. Mt khc, khi chng ti tm tn hiu C22 v C-23 ca -sitosterol l tn hiu ca 2 carbon bc 2 (nhm CH2) th trn ph
DEPT nhn rt r ti 33,34 ppm (C-22) v 25,47 ppm (C-23) (hnh 3.9).

34

Hnh 3.9. Ph 13C-NMR v DEPT ca cht TP-ET-02

Kt hp tt c thng tin chng ti c da trn ph MS-ESI, 1H-NMR,

13

C-

NMR, v DEPT, cng so snh vi d liu ca ph cng hng t ca 2 cht stigmasterol-3-O--D-glucopyranosit v -sitosterol-3-O--D-glucopyranosit [18]
chng ti nhn dng cht TP-ET-02 l hn hp ca -stigmasterol-3-O--Dglucopyranosit v -sitosterol-3-O--D-glucopyranosit.

Hnh 3.10. Cng thc cu to ca -stigmasterol-3-O--D-glucopyranosit

35

Hnh 3.11. Cng thc cu to ca -sitosterol-3-O--D-glucopyranosit

3.2. BAN LUN
Vic nh tnh cc nhm cht hu c trong l cy u rng c thc hin
bng cc phn ng ha hc v sc k lp mng. Kt qu cho thy thnh phn chnh
trong l cy l saponin triterpenoid, ngoi ra cn c cc hp cht sterol, ng kh,
acid hu c v polysaccarid. Kt qu ny khc vi kt qu nh tnh l cy Trevesia
palmata (Roxb. & Lindl.) Vis. thu hi ti Thi Lan ca mt nhm nghin cu ngi
Thi (khng c saponin) [17], tuy nhin li ph hp vi mt nghin cu ti .
Tomasi v cng s phn lp v xc nh c cc saponin trong l cy Trevesia
palmata (Roxb. & Lindl.) Vis. [12].
V chit xut, chng ti kho st dung mi chit l nc v hn hp EtOH
: nc, vi mc ch chit xut c ti a nhom saponin triterpenoid v gim cc
tp cht khc nh chlorophhyll, cht bo, v ng. Kt qu chng ti la chn
c dung mi EtOH 70% chit xut cho cc nghin cu tip theo.
Khi tin hnh chit phn on, chng ti cng s dng phng php chit
thng quy vi cc dung mi co phn cc tng dn. Tuy nhin, trong nghin cu
ny chng ti khng dung dung mi halogen v co c tnh cao vi mi trng v

36

chi ph hy dung mi ny t nn chng ti dng hn hp n-hexan v ethylacetat

t l [5 : 1] co phn cc tng ng vi chloroform.
Cn phn on n-hexan co mu xanh en, dng keo dnh, khi lng t, ch
yu l cc chlorophyll nn ti khng nghin cu tip v phn on ny.
Vi phn on n-hexan/ethyl acetat chng ti th phn tch bng GC-MS
sau khi cn phn on ny c tinh ch qua ct sc k (silica gel) nhm loi b
cht mu. y l ln u tin chng ti th phn tch phn on co phn cc
thp bng GC-MS. V cha co kinh nghim phn tch cc phn on phn cc km
khng phi l tinh du trn h thng my GC-MS nn kt qu thu c trong phn
on ny ch yu l cc alkan, hoc cc cu t phn tch trong mu cha phu hp
vi h thng v th vin ph.
Vi phn on dch chit ethyl acetat, chng ti phn lp c 3 cht bng
sc k ct m. Do lng cht TP-E1-1 v TP-E2-1 t nn chng ti cha o ph
nhn dng cu trc c.
nhn dng cht TP-ET-02, chng ti da trn thng tin ch yu t ph
khi v ph cng hng t ht nhn. trnh lng ph chng ti khng o ngay 1
b ph 1 chiu v 2 chiu m o tng ph mt. Bc u l ph 1H-NMR, chng
ti khai thc ti a thng tin ph cung cp v so snh vi th vin ph gc, v ti
liu tham kho nh hng khung cu trc ri o thm mt s ph khc khng
nh gi thuyt ca mnh l ng. Stigmasterol v -sitosterol (v dng glycosid ca
2 cht ny) thng xut hin trong thin nhin di dng hn hp v rt kh tch.
Trn sc ky khai trin vi nhiu h dung mi khc nhau vn ch cho 1 vt nhng
ph cng hng t proton cho thy cht TP-ET-02 l hn hp ca 2 cht ny dng
glycosid.

37

KT LUN VA KIN NGH

1. KT LUN
Trong khun kh kha lun tt nghip vi ti Nghin cu thnh phn
ha hoc la u rng (Trevesia palmata (Roxb & Lindl) Vis.), chng ti thu
c nhng kt qu sau:
- xc nh c trong l cy u rng (Trevesia palmata (Roxb & Lindl)
Vis.) c cha cc nhm hp cht: saponin triterpenoid, sterol, ng kh, acid hu
c v polysaccarid bng phn ng ha hc.
- kho st 4 h dung mi chit xut l cy u rng v xc nh c
dung mi EtOH 70% l dung mi chit tt nht.
- tin hnh chit xut l u rng bng phng php chit siu m vi
dung mi EtOH 70% v chit phn on vi h dung mi n-hexan/ ethyl acetat
gradient nng .
- phn tch phn on dch chit n-hexan/ethyl acetat [5:1] bng phng
php sc k kh kt hp khi ph GC-MS v so snh vi cc th vin ph cho thy
trong mu phn tch c cha nhiu hp cht alkan.
- phn lp c 3 cht (TP-E1-1, TP-E2-1 v TP-ET-02) bng sc k ct
silicagel pha thng t cn phn on ethyl acetat.
- d on cu trc ca cht TP-ET-02 l hn hp -stigmasterol-3-O--Dglucopyranosit v -sitosterol-3-O--D-glucopyranosit da trn phn tch d liu
cc ph MS-ESI, 1H NMR, 13C NMR, v ph DEPT v so snh vi d liu ph
t ti liu tham kho.
2. XUT
Tip tc phn lp v xc nh cu trc cc cht t cc phn on dung mi
khc ca l cy u rng, c bit dch chit nc cn li sau khi chit 3 phn
on nghin cu.

38

TAI LIU THAM KHAO

TING VIT
1. Nguyn Tin Bn (2003), Danh lc cc loi thc vt Vit Nam. tp 2, NXB
Nng nghip.
2. Vo Vn Chi (2004), T in thc vt thng dng. tp 2, NXB Khoa hc v K
thut.
3. Phm Hong H (2000), Cy c Vit Nam Quyn 2, NXB. Tr, thnh ph H
Ch Minh.
4. L nh Mi, Chu Vn Minh, Trn Vn Sung, Phm Quc Long, Phan Vn
Kim, Trn Huy Thi, Trn Minh Hi, Ninh Khc Bn, L Mai Hng, H
Nhn sm (Araliaceae Juss.)-Ngun hot cht sinh hc v y trin vng Vit
Nam. Hi ngh khoa hoc ton quc v sinh thi v ti nguyn sinh vt ln th 5,
1152-1158.
5. Ng Vn Thu, Trn Hng (2011), Dc liu hoc tp 1, NXB Y hc, H Ni.
6. Nguyn Vn n, Nguyn Vit Tu (1985), Phng phap nghin cu ha hoc
cy thuc, Nh xut bn Y hc, Chi nhnh TP H Ch Minh.
7. Nguyn Vn t, Trn Th Phng Anh (2013), Bc u nghin cu xy dng
khoa nh loi cc chi trong h Ng gia b (Araliaceae) Vit Nam. Hi ngh
khoa hoc ton quc v sinh thi v ti nguyn sinh vt ln th 5, 44 - 46.
TING ANH
8. Abidi S.L. (2001), Chromatographic analysis of plant sterols in foods and
vegetable oils. Journal of Chromatography A. 935(1-2), 173-201.
9. Akihisa T., A.K., Kimura Y., Koike K., Kokke Wcmc, Shibata T., Nikaido T.
(1999), Camelliols A-C, three novel incompletely cyclized triterpene alcohols
from sasanqua oil (Camellia sasanqua). J Nat Prod. 62(2), 265-8.
10. Cheng J. J., Zhang L.J., Cheng H. L., Chiou C. T., Lee I. J., Kuo Y. H. (2010),
Cytotoxic Hexacyclic Triterpene Acids from Euscaphis japonica. J Nat Prod.
11. Committee E. (2007), Flora of China. 13, 436-438.

39

12. De Tommasi Nunziatina, Autore Giuseppina, Bellino Aurora, Pinto Aldo, Pizza
Cosimo, Sorrentino Raffaella, Venturella Pietro (2000), Antiproliferative
Triterpene Saponins from Trevesia palmata. Journal of natural products. 63(3),
308-314.
13. De Tommasi Nunziatina, Bellino Aurora, Pizza Cosimo, Venturella Pietro
(1997), Triterpenoid saponins from Trevesia sundaica. Journal of natural
products. 60(11), 1070-1074.
14. Dinda Biswanath, Debnath Sudhan, Mohanta Bikas Chandra, Harigaya
Yoshihiro (2010), Naturally occurring triterpenoid saponins. Chemistry &
biodiversity. 7(10), 2327-2580.
15. Domingo Victoriano, Arteaga Jess F, del Moral Jos F Qulez, Barrero
Alejandro F (2009), Unusually cyclized triterpenes: occurrence, biosynthesis
and chemical synthesis. Natural product reports. 26(1), 115-134.
16. Jebb, Matthew HP (1998), A revision of the genus Trevesia (Araliaceae).
Glasra. 3(2), 85113.
17. Kannika

Panyaphu,

Panee

Sirisa-ard,

Preeyawis

Na

Ubol,

Surapol

Nathakarnkitkul, Sunee Chansakaow, Tran Van On (2012), Phytochemical,

antioxidant and antibacterial activities of medicinal plants used in Northern
Thailand as postpartum herbal bath recipes by the Mien (Yao) community,
Phytopharmacology. 2(1), 92-105.
18. Khatun Mahbuba, Billah Mirajum, Quader Md Abdul (2012), Sterols and Sterol
Glucoside from Phyllanthus Species. Dhaka University Journal of Science.
60(1), 5-10.
19. Liang Cheng-Qin, Shi Yi-Ming, Li Xing-Yao, Luo Rong-Hua, Li Yan, Zheng
Yong-Tang, Zhang Hong-Bin, Xiao Wei-Lie, Sun Han-Dong (2013),
Kadcotriones AC: Tricyclic Triterpenoids from Kadsura coccinea. Journal of
natural products. 76(12), 2350-2354.
20. Matsuda H., Morikawa T., Ando S., Oominami H., Murakami T., Kimura I.,
Yoshikawa

M.

(2004),

Absolute

stereostructures

of

polypodane-type

40

triterpenes, myrrhanol A and myrrhanone A, from guggul-gum resin (the resin

of Balsamodendron mukul). Chem Pharm Bull (Tokyo). 52(10), 1200-3.
21. Meng Fan-Yu, Sun Jia-Xiang, Li Xue, Yu Heng-Yi, Li Shu-Ming, Ruan Han-Li
(2011), Schiglautone A, a new tricyclic triterpenoid with a unique 6/7/9-fused
skeleton from the stems of Schisandra glaucescens. Organic letters. 13(6),
1502-1505.
22. Takhtajan Armen (2009), Flowering Plants, ed 2, Springer.
23. Verleyen Tom, Forcades M., Verh Roland, Dewettinck Koen, Huyghebaert
Andr, De Greyt Wim (2002), Analysis of free and esterified sterols in
vegetable oils. Journal of the American Oil Chemists' Society. 79(2), 117-122.
24. Vincken Jean-Paul, Heng Lynn, de Groot Aede, Gruppen Harry (2007),
Saponins, classification and occurrence in the plant kingdom. Phytochemistry.
68(3), 275-297.
25. Wen Jun, Lc Phan Ke, Hiep Nguyen Tien, Regalado Jr Jacinto, Averyanov
Leonid V, Lee Chunghee (2007), An unusual new species of Trevesia from
Vietnam and its implications on generic delimitation in Araliaceae. Taxon,
1261-1268.
26. Xu Ran, Fazio Gia C., Matsuda Seiichi (2004), On the origins of triterpenoid
skeletal diversity. Phytochemistry. 65(3), 261-291.

41

PHU LUC
Phu luc 1. Phiu gim nh tn khoa hc v Giy chng nhn m s tiu bn
Phu luc 2. Kt qu phn tch phn on dch chit HE bng phng php GC MS
Phu luc 3. Ph cht TP-ET-02: ph MS negative, MS positive, 1H NMR, 13C NMR
& DEPT

42

PHU LUC 1.
Phiu giam inh tn khoa hoc v Giy chng nhn m s tiu bn

43

44

45

PHU LUC 2.
Kt qu phn tich phn on dich chit HE bng phng phap
GC MS

46

47

48

49

PHU LUC 3. Ph cht TP-ET-02

-

Ph MS negative
Ph MS positive
Ph 1H NMR
Ph 13C NMR & DEPT

50

51

52

53

54

55

56

57

58

59

60