Nghiên cứu các hợp chất có hoạt tính sinh học từ các cây thuốc của Việt Nam

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CAC HOP CHAT CO HOAT TINH SINH HOC TlT
CAC CAY THUdc CUA
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MA SO: QT - 06
-19
CHU TRI
f)t
TAX:
TS PHAN MINH GIANG
;.Al
HGC
QUOC GlA
HA
Npi
|
DT
/

oJ
3
N-
A
HA NOI - 2006
Bao cao torn tit
a. Ten de tai
NghiSn
curu cac hop
ch^t
co hoat tmh sinh hoc tur cac
c^y
thuoc cua Viet
Nam
Ma
s6':
QT-06-19
b.
Chu tri de tai
TS
Phan Minh Giang
c. Cac
sinli
vien tham gia
NCS
Le
Huyen
Tram
ThS Dao Quoc
Himg

CN Nguyen Thi
Tu6
d. Muc tieu va noi dung nghien curu
• Sang lgc
lira
chon mgt so
c^y
thuoc co gia tri su dung cao trong Y hoc
d^n
toe
cua
Vi6t
Nam, giam dinh thuc
vat,
thu hai nguyen
lidu
thuc
vat
dung
cho nghien
cihi.
•
Xtr
ly mSu thuc vat, nghien
cihi
qui
trinh
chiet va phan tach
d^
dieu che

cac phdn chiet va phan doan giau hoat
tinh
sinh hoc.
• Danh gia hoat
tinh
sinh hgc cua cac
phdn
chiet va phan doan.
Dinh hucfng phan tach theo hoat
tinh
sinh hoc.
• Phan tach theo dinh hucfng hoat tfnh sinh hoc. Nghien
cihi
qui trinh phan
lap cac hop
cha^t
co hoat
tinh
sinh hgc bang cac phuong phap phan tfch va
phan tach hien dai.
• Xac dinh
ca'u true
hoa hgc cua cac hop chat ducfc phan lap bang cac
phuang phap vat ly hien dai.
• Danh gia hoat tfnh sinh hgc (chong oxi hoa, khang khuan va khang nam)
cua cac hop
chS^t
dugc phan lap.
• Khao sat ham lugng cac hop cha't co hoat tfnh sinh hoc trong mgl sd cay
thuoc ducfc nghien

cihi.
e. Cac ket qua dat
dirge
1.
Da tien hanh sang lgc lua chgn cay Nghe xanh {Curcuma aff.
aeruginosa Roxb., Zingiberaceae), mgt cay thuoc mdi
dugc
phat hien,
dang duac trong dai tra, va ducfc
sii diing
trong Y hoc dan tgc de dieu
tri cac benh nhu dau
biing
va tang cudng tieu hoa lam doi tugng nghien
ciiu
cua de tai. Da lien hanh giam dinh thuc vat va thu thap nguyen lieu
thuc vat
phue
vu cho nghien
ciiu.
2.
Da
xay
dung dugc cac qui tnnh chiet va phan tach chgn lgc de
di^u
che
cac
phdn chiS't
c6 hoat tfnh sinh hgc,
//-hexan

(hieu suat 3,14% so vai
luong nguyen lieu kho), etyl axetat (0,6%) va
ii-butanol
(0,14%).
3.
Mgt phuong phap phan lap
sac
ky thfch hop da ducfc phat trien de nghien
curu mgt
each
he thong ve hoa hgc va hoat tfnh sinh hgc phan chiet n-
hexan iix than re Nghe xanh. Bang phuong phap nay cac hop cha't thanh
phdn chfnh curdion va zederon da ducfc phan lap v6i cac hieu sua't phan
lap tuong
iing
la 0,1% va 0,05% so vdi lugfng nguyen lieu kho ban ddu.
Phuong phap nay
ciing
thfch hop
d^
phat
tri^n
thanh phuong phap phan
lap lucfng
Idfn
cac hop chdt co hoat tfnh sinh hgc la curdion va zederon,
cung nhu cac che phdm dugfc chiia cac nhom sesquiterpenoid khac nhau.
4.
Cac
kS't

qua nghien
cthi
cac hop chdt de bay hoi cua Nghe xanh da xac
dinh dugc tdng so 23 hgp chdt (chii yeu la cac sesquiterpenoid) vdi
P-elemen
- mgt hgp chdt co hoat tfnh
chdng
ung thu la thanh phdn
chfnh cua cac sesquiterpen hydrocarbon, va curzerenon, curcumol,
germacron va curdion la cac thanh phdn chfnh
ciia
cac sesquiterpenoid
chiia oxy, trong so do curzerenon da dugc sit dung lam thuoc lam sang
cho ung thu da va c6 tur cung.
5.
Cac ket qua nghien
ciiu
cac hgp chdt phan
cue
hon da ddn den viec phan
lap
mirdi
bdn hgp
chS't
tinh
kiiiet tii
than re Nghe xanh, curdion (1), axit
palmitic (2), zederon (3),
y0-sitosterol
(4), stigmasterol (5),

13-hydroxygermacron
(6), aeruginolacton (7), guaianediol (8),
isoprocurcumenol (9), procurcumenol (10), aerugidiol (11), aeruginofuran
(12),
zedoarofuran (13) va aeruginon (14). Nhu vay cac hgp chat dugc
phan lap
CO
cdu tnic ciia cac sesquiterpenoid, cac steroid va axit beo.
Cac sesquiterpenoid aeruginolacton (7) va aeruginofuran (12) la cac hgp
chat huru ca thien nhien mdi, va aeruginon (14) la hgp chat lan dau tien
du*gc phSn
lap
tiT
thien nhien. Cdu tnic hoa hgc ciia cac hgp chdt 1-14 da
dugc xac dinh bang cac phuong phap
ph6 hi6n
dai (UV, IR, MS,
ID
NMR
va 2D NMR). Da sir dung cac phan tfch
ph6
NOED, NOESY va CD trong
xac dinh cac cdu tnic lap thi tuyet ddi cua cac hgp chdt dugc phan lap.
6. Trong cac thu nghiem theo hucfng khang khudn va khang ndm cac phdn
chie't
«-hexan,
etyl axetat va
/i-butanol
da dugc danh gia la co hoat tfnh
khang ndm. Chiing deu ihi hien tac dung khang Aspergillus niger,

Candida albicans va Fusarium oxysporum cf
miic
MIC
tir
25-200
)ag/ml.
Cac phan tach co dinh hudfng khang khudn va khang ndm da ddn den viec
phan lap cac hoat chdt la curdion va zederon.
- Curdion va zederon (cac MIC 25
|ig/ml)
co
th^
dugc phat tridn thanh
cac tac nhan chdng benh thdi re
6
thuc vat gay
bdi
ndm mdc Fusarium
oxysporum.
-
Curdion va zederon da dugc chiing to la cac hgp chdt khang cac vi nam
gay benh cho ngucfi tuong
iing
la Aspergillus niger (MIC 50
|ag/ml)
va
Candida albicans (MIC 25
)ig/ml).
- Curdion (MIC 50
^g/ml),

phdn chiet etyl axetat (MIC 200
(ag/ml)
va
phdn chiet
;j-butanol
(MIC 50
]ig/ml)
co hoat tfnh khang vi khuan
Staphylococcus aureus, Chiing co ihi la nhung ddi tugng nghien
ciiu
thu
vi cho hudfng tim kidm cac hgp chdt co hoat tfnh khang Staphylococcus
aureus khang methicillin (MRSA) va vancomycin (VRSA).
7.
Da phan lap va xac dinh dugc cac hgp chdt co hoat tfnh bao
\t
gan trong
than
rS
Nghe xanh nhu furanodien, germacron, curdion, 13-
hydroxygermacron
va
aerugidiol. Furanodien
va
13-hydroxygermacron
cung
la cac hgp
chdt
da
dugc chiing

td la co tac
dung
lie
che'
sir
san
sinh
cua nito oxit (NO).
8. Trong
cac
thur nghiem
v^
hoat tfnh chdng
oxy hoa (dgn gdc tu do) va
gay
dgc te'
bao ddi vdi ba
ddng
te' bao ung thu
ngudi
la
Hep-Gj,
Fl va RD, ca
curdion
va
zederon
diu
khong
(hi
hien

cac
hoat tfnh dang
chu y.
9.
Cac ke't qua
ciia
De tai da ddn den
sir
chuan
bi
cdng
bd hai bai bao
tren
cac
tap chf
ciia Viet
Nam
Duac
hpc
va
qudc
te'
Chemical
and
Pharmaceutical Bulletin.
Mgt
sinh vien
da tdt
nghiep
va ba

sinh vien
nam
thur
3 da
hoan thanh nghien
ciiu
khoa
hgc
trong khudn
khd
ciia
de tai nay.
f.
Tinh hinh
kinh
phi cua de tai
Tdng kinh
phf
dugc cdp: 15.000.000 ddng
Cac khoan
chi
cila
de tai da
dugc phan
bd nhu sau:
Muc
(%)
109
110
114

119
Npi
dung chi
Thanh toan djch vu cong cong
Vat tu van phong
Thue thiet bi cac loai
(thue thie't bi ghi do phd,
thir
hoat tfnh sinh hoc)
Chi
phi
nghiep vu chuyen mon
(Nguyen lidu, hoa
ch^t,
dung cu)
Quan ly
phi
Tdng c6ng
So tien (dSng)
600.000
873.000
6.310.000
6.617.000
600.000
15.000.000
Tyle
4,0
5,8
42,1
44,1

4,0
100.0
KHOA QUAN
Lt
(Ky va ghi ro ho ten)
CHU TRI
Dt
TAI
(Ky va ghi ro ho ten)
Phd Chu nhiem Khoa Hoa hoc
PGS.
TS Trdn Thi
Nhir
Mai
il.
'iim
TS Phan Minh Giang
TRlTCfNG
DAI HOC KHOA HOC
Tl/NHlfiN
PHOHI^UTRUONU
PGSJS.^Aw^iu^am.
Outline Report
a. Project title
Study on biologically active compounds from Vietnamese medicinal
plants
Code
No.: QT-06-19
b.
Head of the project

Dr Phan Minh Giang
c. Participating researchers
PhD student Le Huyen Tram
MSc Dao Qudc Hung
BSc Nguyen Thi Tue
d.
Objectives and main contents of the project
• To carry out selection, taxonomic identification and collection of some
interesting medicinal plants of Vietnam for the research.
• To perform stabilization of the plant materials, extraction and
fractionation procedures for the preparation of biologically active extracts
and fractions.
• To evaluate biological activities of the extracts and fractions.
To perform activity-guided fractionation of the extracts and fractions.
• To study isolation procedures of biologically active compounds using
modem analytical and separation methods.
• To determine structures of the isolated compounds on the basis of modem
physical methods.
• To evaluate biological (antioxidant, antibacterial, antifungal, and
cytotoxic) activities of the isolated compounds .
• To examine contents of the biologically active compounds in the
medicinal plants studied.
e. Study results
1.
Curcuma aff. aeruginosa Roxb., Zingiberaceae (Vietnamese name: Nghe
xanh),
a medicinal plant, which was recently discovered and widely
cultivated for the use in the treatment of stomach ache and in the
enhancement of digestion, was selected for the study. The plant was
taxonomically identified and collected for the study.

2.
A selective extraction and fractionation procedure was established for the
preparation of the biologically active n-hexan (3.14% yield on the basis of
the dry material), ethyl acetate (0.6%) and
/i-butanol
(0.14%) extracts.
3.
A suitable chromatographic isolation method was developed for the
systematic chemical and biological study of the
/z-hexan
extract of the
rhizomes of C. aff. aeruginosa. By using this method the main
constituents curdion and zederon were isolated in the yields of 0.1% and
0.05%,
respectively, of the dry material. This method will be suitable for
the development of large-scale isolation of the biologically active curdion
and zederon, as well as pharmaceutical preperations containing different
groups of sesquiterpenoids.
4.
The study of the volatile compounds of C. aff. aeruginosa determined in
total 23 compounds (mainly sesquiterpenoids), of which
P-elemene
- an
anticancer compound - was the main constituent of the sesquiterpene
hydrocarbons, and curzerenone, curcumol, germacrone, and curdione
were the main components of the oxygenated sesquiterpenoids, of which
curzerenone was used as a clinical drug for cervix and skin carcinomas.
5.
The study of the more polar compounds led to the isolation of fourteen
pure compounds from the rhizomes of C. aff. aeruginosa, curdione (1),

palmitic acid (2), zederone (3),
y3-sitosterol
(4), stigmasterol (5),
13-hydroxygermacrone
(6), aeruginolactone (7), guaianediol (8),
isoprocurcumenol (9), procurcumenol
(10),
aerugidiol (11), aeruginofuran
(12),
zedoarofuran (13), and aeruginone (14). The isolated compounds
possess the structures of sesquiterpenoids, steroids, and fatty acid.
The sesquiterpenoids aeruginolactone (7), aeruginofuran (12) are new
organic natural compounds, and aeruginone (14) was isolated from
Nature for the first time. The chemical structures of compounds 1-14 were
determined by using modem spectroscopic methods (UV, IR, MS,
ID
NMR, and 2D NMR). NOED, NOESY, and CD spectra were used in the
determination of the absolute stereostructures of the compounds isolated.
6. In the antibacterial and antifungal assays n-hexan, ethyl acetate, and
n-butanol extracts showed inhibitory activity against Aspergillus niger,
Candida albicans and Fusarium oxysporum at
MICs
of 25-200
(ag/ml.
Activity-guided fractionation led to the isolation of the bioactive
compounds curdione and zederone.
- Curdione and zederone (MICs of 25
|ag/ml)
can be developed into
agents which were active against the blockage and breakdown of

xylem
of plants caused by Fusarium oxysporum.
- Curdione and zederone were demonstrated to be active against the
human pathogenic fungi Aspergillus niger (MIC of 50
^g/ml)
and
Candida albicans (MIC of 25
|ig/ml),
respectively.
- Curdione (MIC of 50
^g/ml),
ethyl acetate (MIC of 200
^ig/ml),
and
n-butanol (MIC of 50 fig/ml) extracts showed inhibitory effect against
Staphylococcus aureus. They can be selected for the study toward the
discovery of active compounds against methicillin-resistant
Staphylococcus aureus (MRSA) and vancomycin-resistant (VRSA)
activities.
7.
The hepatoprotective compounds were isolated and identified in the
rhizomes of C. aff. aeruginosa such as furanodiene, germacrone,
curdione,
13-hydroxygermacrone,
and aerugidiol. Furanodiene and
13-hydroxygermacrone
were demonstrated to inhibit the nitric oxide (NO)
production.
8. In the antioxidant (radical scavenging activity) and cytotoxic activity
against the human cancer cell lines

Hep-Gj,
Fl, and RD bioassays
curdione and zederone did not show noticeable effects.
9. The project resulted in the preparation of two publications in the national
Vietnam Pharmaceutical Journal and the intemational Chemical and
Pharmaceutical Bulletin. One BSc student graduated and three
3'**
year
students completed the Research
training
in the framework of the project.
Muc luc
Trang
Muc luc cac chii vie't tat 1
1 Ldi md ddu 2
2 Ndi dung chfnh 3
2.1 Phuong phap nghien
cihi
3
2.2 Phuong phap va thiet bi 5
2.3 Nguyen lieu thuc vat 5
2.4
Di^u
che'
cac phdn chiet
tir
than
re
Nghe xanh (C. aff.
aeruginosa)

5
2.5 Phan tach phdn chiet n-hexan va phan lap cac sesquiterpenoid 6
2.6 Phuong phap
thii
hoat
tinh
sinh hoc 6
2.6.1
TTiir
hoat tfnh khang khudn va khang ndm in vitro 6
2.6.2 Thijf hoat tfnh chdng oxy hoa in vitro 6
2.6.3
Hiir
hoat tfnh gay ddc
te'
bao in vitro 6
2.7 Hdng sd vat ly va du kien phd cua cac hgp chdt dugc phan lap 7
2.8 Ket qua va Thao luan
11
2.8.1 Phan lap va xac dinh cdu tnic cac sesquiterpenoid
tir
Nghe xanh
11
(C.
aff.
aeruginosa,
Zingiberaceae)
2.8.2 Danh gia cac hoat tfnh sinh hoc cua Nghe xanh 22
(C.
aff. aeruginosa, Zingiberaceae)

3.2.1 Hoat tfnh khang khudn va khang ndm in vitro 22
3.2.2 Hoat tfnh chdng oxy
hoa/n
v/rr<9
24
3.2.3 Hoat tfnh gay ddc
te'
bao in vitro 24
3.
Ke't luan 24
4.
Tai lieu tham khao 26
Phu luc
MUC LUC CAC
CH0 VI^T
TAT
TLC:
Tliin
layer chromatography
CC:
Open column chromatography
FC:
Rash chromatography
ODS:
Octadecyl silica gel
MPLC:
Medium pressure liquid chromatography
HPLC:
High performance liquid chromatography
GC:

Gas chromatography
GC-MS: Gas chromatography - Mass spectroscopy
UV: Ultraviolet spectroscopy
IR: Infrared spectroscopy
NMR: Nuclear magnetic resonance
ID
NMR: One dimenssional nuclear magnetic resonance
2D NMR: Two dimenssional nuclear magnetic resonance
^H-NMR:
Proton nuclear magnetic resonance
^^C-NMR:
Carbon 13 nuclear magnetic resonance
DEPT: Distortionless enhancement by polarization transfer
NOE:
Nuclear Overhauser effect
NOED: Nuclear Overhauser effect difference spectroscopy
NOESY: Nuclear Overhauser effect spectroscopy
'H-^H
COSY: Proton - proton correlation spectroscopy
HMQC:
Heteronuclear multiple quantum correlation
HSQC:
Heteronuclear single quantum correlation
HMBC:
Heteronuclear multiple bond correlation
CD:
Circular dichroism
1
Ldl MCJ
DAU

Thuc vat la nguon cung cdp cac hgp chdt thien nhien cd cau tnic hoa hoc va
hoat tfnh sinh hgc rdt da dang. Cac qua
trinh
chiet xuat, phan lap, va xac dinh
cdu tnic hoa hoc va hoat tfnh sinh hoc
tir
cac thuc vat dung lam thudc trong Y
hgc dan tdc ddn den
sir
phat hien cac loai dugc pham mdi cd tac dung dieu tri
cao.
Theo Farhsworth N. R. [1] cd ft nhdt
119
hgp chdt dan xudt tir 90
loai
thuc
vat dugc
sir
dung lam cac dugc phdm quan trgng trong dieu tri. Mot trong nhimg
ddn chiing kinh dien la aspirin dugc ddn xudt tir axit salixylic tir la va vd cay
Lieu
(Salix
alba L., Salicaceae) hoac la taxol, mdt trong nhung thudc dieu tri
ung thu quan trong trong hai thap ky cudi, tir cay Thdng Thai
Binh
Duong
(Taxus brevifolia Nutt., Taxaceae). Cac nghien
cihi
trong
ITnh

vuc hien nay tap
trung vao viec phat hien cac hgp chdt
"ddn
dUcmg'
cd hoat tfnh sinh hpc cao tir
nguon thien nhien va tdi uu hoa cac hgp chdt
''ddn
ducrtig'
tir thien nhien su
dung cac phuong phap
chuye'n
hoa hoa hgc. Mot sd nghien
ciiu
gdn day
ciia
chiing tdi da phat hien dugc cac
enf-kauran
diterpenoid tir cay Khd
sam
cho
la
{Croton tonkinensis Gagnep., Euphorbiaceae) va chalcon cardamomin tir cay
Rieng rirng {Alpinia conchigera
Griff.,
Zingiberaceae)
th^
hien cac tac dung
chdng viem manh thdng qua viec
lic
che yeu td phien ma

NF-KB
(nuclear factor
kappa B) va s,U san sinh cua nito oxit (NO)
[2,3].
Cac
^nr-kauran
diterpenoid
dugc phan lap tir cay Khd sam cung ddng thdi dugc chiing
id la
cac hgp chat co
hoat tfnh chdng ky sinh trung sd ret Plasmodium falciparum [4],
gay
doc ddi vdi
Artemia salina [5], khang khuan va khang nam, dac biet la vi khuan
Staphylococcus aureus
[6].
Dieu nay cho thdy trien vong tie'p tuc md rdng hudng
nghien
ciiu
nay cho cac nhdm hgp chdt thien nhien khac tir cac cay thudc khac
cua Viet Nam va vdi nhiing hoat tfnh sinh hgc mdi.
Cac
cay
thudc ho Gimg (Zingiberaceae), bao gdm cac chi nhu
Alpinia,
Amomum, Curcuma, Hedychium, Kaempferia, va Zingiber [7] dugc
sii
dung
phd bien
d^

lam thudc va thuc pham. Y hoc dan tdc Viet Nam thudng sir dung
cac loai Nghe {Curcuma) nhu Nghe vang {Curcuma longa L.), Nghe den
[Curcuma zedoaria (Berg.) Roscoe], Nghe
re
vang {Curcuma
xanthorrhiza
Roxb.),
Nghe ten ddng {Curcuma aeruginosa Roxb.) va Nghe udng {Curcuma
aromatica Salisb.) trong dieu tri cac benh ung thu, viem loet, xO gan va an udng
khdng tieu. Trbng mot chuong
Uinh
tie'p tuc nghien
ciiu
ve cac hgp chat cd hoat
tfnh sinh hgc i\s cac cay thudc dan tdc Viet Nam, Nghe xanh {Curcuma aff.
aeruginosa Roxb.), mdt loai Nghe mdi dugc phat hien, da dugc
lira
chon
lam
ddi
tugng nghien cuu ve hoa hgc va hoat tfnh sinh hoc.
Nghe xanh (C. aff. aeruginosa Roxb.) la loai cay thao cao tir 1-2 m. Vc
mat thuc vat hgc cay Nghe xanh tuong tu nhu cay Nghe ten dong (C.
aeruginosa Roxb.) [8]. Tuy nhien, didm khac biet giua Nghe xanh va Nghe
ten ddng la
Idt
cdt ciia than rl Nghe xanh thdm hon cua Nghe ten ddng, hoi
nga sang mau xanh muc Ciru Long. Hien nay cay dang dugc trdng d Ha Ndi,
Vmh Phiic va Hai Phdng di thu than
re

lam thudc chira cac benh nhu dau
bung va tang cudng tieu hoa [8].
De tai da tie'n hanh
xay
dung cac phuong phap chiet va phan tach sdc ky de
thuc hien cac nghien
cihi
cd he thdng ve thanh phdn hoa hgc va hoat tfnh sinh
hgc ciia Nghe xanh {Curcuma aff. aeruginosa Roxb., Zingiberaceae). Cac
hctp
chdt thanh phdn da dugc phan lap va xac dinh cdu tnic hoa hoc bang cac phuong
phap phd hien dai. Cac hoat tfnh sinh hgc (khang khudn, khang nam, chdng oxy
hoa va gay dgc te' bao) cua mdt sd hgp chdt thanh phdn quan trgng da dugc danh
gia trong mgt mdi tuong quan vdi hoat tfnh sinh hgc
ciia
cac phdn chiet va tac
dung
chira
benh trong Y hgc dan tdc cua Nghe xanh. Nhimg ket qua nghien
ciiu
mdi cua
66
tai nay da ddng gdp vao viec xay dung cac kien thirc vl hoa hgc va
dugc ly hgc cua
cay
Nghe xanh.
2 NOI DUNG CHINH
2,7
Phuomg phdp nghien cicu
Ddi tugng nghien

ciiu
cua de tai la
cay
Nghe xanh {Curcuma aff. aeruginosa
Roxb.,
Zingiberaceae) dugc
lira
chgn
uen
co sd tac dung chira benh Uong Y hpc
dan tdc ciia Viet Nam. Cac loai Nghe {Curcuma) thupc hp Zingiberaceae thudng
dugc
sir
dung mdt
each
an toan trong thuc pham va Y hgc dan tpc do dd cac hgp
chdt cd hoat tfnh sinh hpc dugc phat hien tir cac loai nay se cd
trie'n
vpng phat
tridn
thanh cac tac nhan dieu
Ui
cac loai benh khac nhau.
Phuong phap nghien ciru he thdng ve hoa hpc dugc bdt dau bdng chiet chpn
lpc cac ldp chdt chfnh
sir
dung cac dung mdi hiiu co theo thu' tu dp phan
cue
tang ddn. Mpt phuong phap phan tach cac phdn chiet dugc phat trien tren co sd
cac khao sat sdc ky; phuong phap phan tach nay cho phep gpp cac hgp chdt

hiru
cO cd cdu tnic lien quan thanh cac nhdm hgp chdt khac nhau (cac nhom phdn
doan).
Dieu nay cho phep ddy nhanh cac qua trinh sang lpc
ve
hoa hpc va hoat
tfnh sinh hpc,
sir
dung cac ky thuat sdc ky hien dai nhu sdc ky Idp mong (TLC),
sdc ky khf (GC), sdc ky ldng hieu nang cao (HPLC) phan tfch va dac biet
la
cac
ky thuat ghep ndi {hyphenated technique) nhu sdc ky khf - khd phd ke
lien
hop
(GC-MS). Cac nhdm phan doan nay dugc tiep tuc phan tach bdng cac ky thuat
sdc ky hien dai nhU sdc ky cot thudng (CC), sdc ky cdt nhanh (FC), sdc ky Idng
trung dp (MPLC), sdc ky long hieu nang cao (HPLC) dieu che de cho cac hgp
chat tinh khiet.
Cdu
true
hoa hgc cua cac hgp chdt dugc phan lap dugc xac dinh bdng cac
phuong phap phd hien dai nhu phd tii ngoai (UV), phd hdng ngoai
(IR),
phd
khdi lirgng va cham dien
tii
(EI-MS), phd khdi lugng bdn pha nguyen tir nhanh
(FAB-MS) phan giai cao, cac phd cdng hudng tir hat nhan mot chieu
(ID

NMR)
va hai chidu (2D NMR) vdi cac ky thuat khac nhau. Cdu tnic lap thd mong ddi
va tuyet ddi ciia cac hgp chdt nay dugc xac dinh bdng phan tfch cac phuong
phap phd nuclear Overhauser effect mdt chieu (NOED) hoac hai chieu
(NOESY) va phd circular dichroism (CD).
Cac phdn chiet dugc thir nghiem cac hoat tfnh sinh hgc nhdm dinh vi cac
hgp chdt cd hoat tfnh sinh hpc cdn dugc xac dinh. Cac hgp chdt tinh khiet dugc
phan lap dugc ihix nghiem mgt sd hoat tfnh sinh hgc (hoat tfnh khang khuan va
khang ndm, chdng oxy hoa va gay ddc te' bao) trong mpt mdi tuong quan vdi
hoat tfnh cua cac phdn chie't va tac dung dugc ly hgc dan tpc cua
cay
thudc
dugc nghien ciru.
Phuong phap nghien
cixu
nay dugc tdm tdt trong So dd
1.
So do 1. Qui trmh phan lap cac hgp chat co hoat tinh sinh hgc tir
Nghe xanh (Curcuma aff.
aeruginosa^
Zingiberaceae)
Curcuma aff. aeruginosa Roxb.
(Zingibearceae)
I
Cac phdn chiet
c
phan tich sac ky
hoat
tinh
sinh hoc

)
Cac nhom phan doan
-<-
)
CC,FC,
MPLC, HPLC
Cac hcrp chat tinh khiet
UV, IR, MS, NMR, CD
Cau
true
hoa hoc
2.2 Phuorng phdp vd thiet bi
Didm ndng chay (dnc) dugc do tren thiet bi Boetius va khdng dugc hieu
chinh. Do quay
cue {[a]^)
dugc do
tren
may JASCO P-1030 digital polarimeter.
Phd
tii
ngoai (UV) dugc do tren thiet bi GBC Instrument 2855 spectrometer. Phd
hdng ngoai (IR) dugc do tren cac may Impact-410-Nicolet FT-IR
spectrophotometer va Horiba FT-710 spectrophotometer. Phd circular dichroism
(CD) dugc do tren may JASCO J-720 spectropolarimeter. Phd khdi luong va
cham dien
tii
(EI-MS) dugc do tren may HP-5989 B MS. Phd HR-FABMS ion
am
dugc do tren may JEOL SX-102 mass spectrometer vdi matrix chudn PEG-
400.

Phd cdng hudng tir proton
(*H-NMR)
va cacbon 13
(^^C-NMR)
dugc ghi
tren cac may Bruker
AV
500 spectrometer va JEOL
JNM-a
400 NMR
spectrometer.
Sdc ky ldp mdng (TLC) dugc thuc hien tren ban mdng Uang sdn Merck
Alufolien DC 60
F254.
Cac vet chdt dugc phat hien bang
each
phun thudc thir
10%
H2SO4
trong 50% EtOH hoac
vanilin/H2S04
dac 1% sau dd ho ndng d
200°C,
va bang den tir ngoai d budc sdng
X
254 nm. Chdt hdp phu
dimg
cho sdc
ky cdt la silica
gel

Merck (cd hat 63-200
jam)
va chdt hdp phu pha dao YMC
octadecyl
siHca
gel (ODS). Sdc ky ldng hieu nang cao (HPLC) phan tfch va dieu
che'
dugc thuc hien d budc sdng
A,
210 nm, sir dung cdt YMC ODS (kfch thudc
150
X
4,6 mm dudng kfnh trong cho phan tfch va 150 x 20 mm dudng kfnh
trong cho dieu
che),
vdi cac tdc do ddng tuong img la 0,5 va 5
ml/phiit.
Sdc ky
khf - khdi phd ke
lien
hgp (GC-MS) dugc thuc hien tren thiet bi GC HP 5890
II
sur dung cdt mao quan HP-5 (30 m x 0,25 mm dudng kfnh trong, ldp phim day
0,25
^m)
lien hgp vdi MSD 5989 B.
2.3 Nguyen lieu thuc vgt
Than
re
tuoi Nghe xanh (Curcuma aff. aeruginosa Roxb., Zingiberaceae)

dugc thu thap d
Phii
TTiuy,
Gia Lam, Ha Ndi.
20 kg than rl nghe xanh tuoi dugc rira sach, thai lat mdng, phoi khd trong
bdng dam, sdy d 45-
50°C,
rdi xay thanh bdt min, thu dugc 5 kg bdt khd.
2.4 Dieu che cdc phdn chiet
tit
thdn ri Nghe xanh
(C.
aff. aeruginosa)
Bdt than
re
Nghe xanh khd (5 kg) dugc ngam chiet trong metanol 6 Idn
6
nhiet do phdng, mdi ldn trong 3 ngay. Sau khi lgc bd ba, dich chiet metanol
dugc cdt loai dung mdi dudi ap sudt giam xudng cdn 1/10 the tfch, roi pha
loang vdi nudc cdt. Dich
metanol-nudc
dugc chiet chgn lpc vdi n-hexan,
EtOAc va n-butanol, cac dich chie't dugc lam khan bdng
Na2S04,
sau dd dugc
cdt loai dung mdi cho cac phdn chiet tuong
iing
la phdn chie't n-hexan (157 g,
hieu sudt 3,14% so vdi lugng nguyen lieu khd). etyl axetat (30 g, 0,6%) va
n-butanol

(7
g,
0,14%).
2.5
Phdrt
tdch phdn
chie't
n-hexan
vd phdn lap cdc sesquiterpenoid
83 g phdn chie't n-hexan dugc tien hanh phan tach bang CC tren silica
gel,
nia giai gradient vdi n-hexan va cac he dung mdi n-hexan-EtOAc
(19:1,
9:1,
4:1,
2:1 va 1:1). Dua tren thd tfch ciia cac he dung mdi rira giai cac phan doan
dugc gdp lai va phan tfch bdng TLC, ke't qua la thu dugc tam nhdm phan doan,
nhdm 1
(CAl-2),
nhdm 2 (CA3-7), nhdm 3 (CA8-9), nhdm 4
(CAlO-15),
nhdm
5 (CA16-20), nhdm 6 (CA21-25), nhdm 7 (CA26-27) va nhdm 8
(CA28-31)
(So dd 2). TCr 5 g nhdm phan doan 2 (45,4 g), bdng CC tren silica gel vdi he
dung mdi
n-hexan-EtOAc
(25:1), va ke't tinh lai trong n-hexan, cho 300 mg
curdion (1) va 10 mg axit palmitic (2). Tir 10 g nhdm phan doan thii' 4
(14,1

g),
bang CC tren silica gel vdi he dung mdi n-hexan-EtOAc (13:1 va 9:1) va kcl
tinh lai trong n-hexan cho 1,65 g zederon (3) va 40 mg hdn hgp cua
/?-sitosterol
(4) va stigmasterol (5). Nhdm phan doan 5 (3,1 g) dugc phan tach bdng sdc ky
cdt CC tren ODS
(MeOH-H20,
3:2
va
7:3) sau dd dugc tinh che bdng HPLC
dieu che' tren cdt ODS
(MeOH-H20,
7:3) dd cho
13-hydroxygermacron
(6)
(53,9 mg) va aeruginolacton (7) (21,2 mg). Nhdm phan doan 6 (4,0 g) dugc
phan tach bdng CC tren ODS
(MeOH-H20,
3:2 va 7:3) va
ti^p
tuc bdng HPLC
dieu che'
uen
cot ODS
(MeOH-H20,
3:2) dd cho guaianediol (8) (18,2 mg),
isoprocurcumenol (9) (42,3 mg) va procurcumenol (10) (95,7 mg). Nhdm phan
doan 7 (1,7 g) va 8 (1,7 g) dugc phan tach bdng CC tren ODS
(MeOH-H.O,
3:2

va 7:3) va HPLC didu che tren cdt ODS
(MeOH-H^O,
1:1) dd cho aerugidiol
(11) (96,3 mg), aeruginofuran (12) (2,1 mg), zedoarofuran (13) (21,0 mg), va
aeruginon (14) (19,1 mg).
2.6 Phuang phdp
thii
hogt tinh sinh
hpc
2.6.1
Thic
hogt tinh khdng khudn vd khdng
ndm
in vitro
Hoat tfnh khang khuan va khang ndm dugc danh gia so bd va ndng dp
lic
che tdi thidu(MIC) dugc xac dinh vdi cac mdu thd hien hoat tfnh khang khudn va
khang ndm bang phuong phap vi pha loang
tren
phien 96 gieng [9j. Cac khang
sinh kidm dinh bao gdm ampixilin [ddi vdi vi khudn Gram (+)],
tetracylin
[ddi
vdi vi khuan Gram
(-)]
va nystatin (ddi vdi ndm sgi va ndm men).
2.6.2
Thii
hogt tinh chdng oxy hod in vitro
Hoat tfnh chdng oxy hoa dugc danh gia bang phuong phap dpn gdc 2,2-

diphenyl-1-picrylhydrazyl (DPPH) trong dung dich etanol bao hoa [10].
2.6.3
Thic tdc
dung gdy
dgc
tebdo in vitro
Tac
diing gay
ddc te' bao dugc
thii
theo phuong phap dang dugc thuc hicn tai
Vien nghien
cihi
ung thu Qudc gia Hoa Ky (National Cancer Institute, NCI)
[11] vdi ba ddng te' bao ung thu ngudi la
Hep-G2
(ung thu gan),
R
(ung thu
mang tir cung) va RD (ung thu mang tim).
2.7 Hang so
vdt
ly vd
du:
kien
phd
cua cdc hop
chdt
duac phdn lap
Curdion (1):

Tinh
th^
hinh
kim, mau trang, dnc
60-62°C,
[a]"+26,0° (c
1,0,
CHCI3).
Rf=
0,38 (TLC, silica gel,
/i-hexan-EtOAc,
9:1).
IR (KBr):
v„„
1696, 1665
cm^.
EI-MS:
m/z 236
(C.jHj^O^,
M^).
'H-NMR
(CDQj):
5 0,88 (3H, d,
/
= 6,7 Hz,
I2-CH3),
0,95 (3H, d,
7 =
6,6 Hz,
13-

CH3),
0,98 (3H, d,
7
= 7,0 Hz,
I5-CH3),
1,58
(IH,
dddd,
7
= 4,1 Hz, 3,2 Hz, 3,1 Hz,
3,0 Hz, Ha-3), 1,66 (3H, s,
I4-CH3),
1,88
(IH,
dqq, J
=
2,1 Hz, 6,6 Hz, 6,7 Hz, H-
11),
2,07-2,16 (3H, m, 2H-2, Hp-3), 2,34
(IH,
dddd, J = 7,6 Hz, 7,1 Hz, 4,1 Hz,
2,2 Hz, Ha-4), 2,39
(IH,
dd, J = 14,0 Hz, 2,2 Hz, Hp-6), 2,69
(IH,
dd,
/
= 8,4 Hz,
8,0
Piz,

Ha-6), 2,85
(IH,
ddd, J = 7,0 Hz, 6,9 Hz, 1,9 Hz, Hp-7), 2,93
(IH,
d,./ =
10,9 Hz, Ha-9), 3,09
(IH,
d,y
= 10,9 Hz, Hp-9), 5,16
(IH,
d,./
=
8,1 Hz,, H-l).
"C-NMR
(CDCI3):
5 16,6 (q, C-15), 18,5 (q, C-14), 19,8 (q, C-12), 21,1 (q, C-13),
26,4 (t, C-2), 29,9 (d,
C-11),
34,0 (t, C-3), 44,2 (t, C-6), 46,8 (s, C-4), 53,4 (d, C-7),
55,9 (t, C-9), 129,9 (s, C-10), 131,5 (d, C-l),
211,1
(s, C-5), 215,0 (s, C-8).
Axit palmitic (2):
Tinh
\h6 hinh
kim, khong mau, dnc
52-54"C.
Rf
= 0,6 (TLC, silica gel,
/j-hexan-ElOAc,

9:1).
IR (KBr):
v„,,
3500-2500, 1703 cm'.
EI-MS:
m/z 256
{C,^U,^0„
W).
Zederon (3):
Tinh
the'
hinh kim, mau trang, dnc
150-152°C,
[a]"+265,8°
(r 1,0, CHCI3).
Rf
= 0,77 (TLC, silica gel,
/i-hexan-EtOAc,
4:1).
IR (KBr):
v„3,
1698, 1457, 1275, 1056, 921 cm'.
EI-MS:
m/z 246
(Ci^Hi^Oj,
M^).
'H-NMR
(CDCI3):
5
1,29

(IH,
ddd,
/
= 9,6 Hz, 9,0 Hz, 4,1 Hz, Hp-3), 1,34
(3H, s,
I5-CH3),
1,60 (3H, s,
I4-CH3),
2,10 (3H, br s,
I3-CH3),
2,24
(IH,
br d,
J
= 7,8 Hz, HP-2), 2,29
(IH,
ddd,
/ =
6,1 Hz, 3,5 Hz, 3,4 Hz, Ha-3), 2,53
(IH,
dddd, J = 8,4 Hz, 8,3 Hz, 3,5 Hz, 2,0 Hz, Ha-2), 3,68
(IH,
d,
7 =
16,4 Hz, Ha-
9),
3,75
(IH,
d, 7
=

16,4 Hz, Hb-9), 3,81
(IH,
s, H-5), 5,48
(IH,
m, H-l), 7,08
(IH,
brs, H-12).
"C-NMR
(CDCI3):
6 10,2 (q, C-13), 15,1 (q, C-15), 15,7 (q, C-14), 24,6 (t, C-2),
37,9 (t, C-3), 41,8 (t, C-9), 63,9 (s, C-4), 66,5 (d, C-5), 122,1 (s, C-10), 131,0 (s, C-
7),
123,2 (s, C-11), 131,1 (d, C-l), 138, d (C-12), 157,0 (s, C-8), 192,1 (s, C-6).
H6n hop cua p-sitosterol va stigmasterol (4/5):
Tinh
th^
hinh kim, mau tring,
di^m
nong chay
134-136°C.
Rf
= 0,43 (TLC, silica gel,
«-hexan-etyl
axetat, 9:1).
IR
(KBr):
v^^
3434,
1648, 1380, 1050, 1022,
958 cm'.

EI-MS:
m/z 414
(3,
Q^HjoO,
M*),
412
(4,
C29H50O,
M^).
'H-NMR (CDQj)
(3):
8
0,68
(3H, s,
I8-CH3),
0,91
(3H,
d,
7 =
6,6
Hz,
21-CH,),
0,81
(3H, d,
7 =
6,9
Hz,
26-CH3),
0,83
(3H, d,

7 =
7,3
Hz,
27-CH3),
0,84
(3H,
t,
7
=
7,6
Hz,
29-CH3),
3,51
(IH,
tt,
7 =
11,7
Hz,
5,1
Hz, H-3),
5,35
(IH,
br
d,
7 =
5,1
Hz,
H-6),
1,0
(3H,

s,
I9-CH3).
'H-NMR (CDQj)
(4):
5
0,70
(3H,
s,
I8-CH3),
0,79
(3H, d,
7 =
7,0
Hz,
26-CH3),
0,80
(3H,
t,
7 =
7,7
Hz,
29-CH3),
0,84
(3H, d,
7 =
7,1
Hz,
27-CH3),
1,0
(3H,

d,
7
=
6,5
Hz,
2I-CH3),
1,0
(3H, d,
7 = 6,5
Hz,
I9-CH3),
3,51
(IH,
tt,
7
= 11,7
Hz,
5,1
Hz, H-3),
5,35
(IH,
br
d,
7
=
5,1
Hz,
H-6),
5,0
(IH,

dd,
7 =
14,4
Hz,
9,0 Hz,
H-23),
5,15
(IH,
dd,
7 =
14,4 Hz,
9,0
Hz, H-22).
"C-NMR (CDa3)
(3):
8
11,9
(q, C-18),
12,0
(q, C-24),
18,8
(q, C-21),
19,0 (q,
C-26),
19,4
(q, C-19),
19,8
(q, C-27),
21,1
(t, C-11),

23,1
(t, C-28),
24,3
(t,
C-
15),
26,1
(t, C-23),
28,2
(t, C-16),
29,1 (d,
C-25),
31,4
(t, C-7),
31,7
(t,
C-2),
31,9
(d, C-8),
33,4
(t,
C-22),
39,8
(t,
C-12),
36,2
(d, C-20),
36,5
(s, C-10),
37,3

(t,
C-l),
42,3
(s,
C-13),
45,8
(d, C-24),
50,1
(d, C-9),
56,1
(d, C-17),
56,8
(d,
C-
14),
71,8
(d,
C-3),
121,7
(d,
C-6),
140,8
(s,
C-5).
13-Hydroxygermacron
(6):
Bot v6 dinh hinh.
'H-NMR
(CDCI3):
8 1,38 (3H, s,

I5-CH3),
1,58 (3H, s,
I4-CH3),
1,76 (3H, s.
I2-CH3),
2,92
(IH,
d, 7 = 10,8 Hz, Ha-9), 3,81
(IH,
d, 7 = 10,8 Hz, Hb-9),
4,12(1H, d,7=
12,4Hz,
Ha-13),
4,23
(IH,
d, 7= 12,4 Hz,
Hb-13),
4,62
(IH,
br d, 7 = 6,8 Hz, H-l), 4,93
(IH,
br d, 7 =
11,2
Hz, H-5).
"C-NMR
(CDCI3):
8 16,1 (q, C-15), 17,2 (q, C-14), 18,3 (q, C-12), 24,6 (t, C-2),
29,1 (t, C-6), 38,6 (t, C-3), 56,0 (t, C-9), 63,2 (t, C-13), 125,6 (d, C-5), 126,9 (s, C-
4),
132,0 (s, C-7), 133,6 (d, C-l), 136,1 (s, C-10), 140,2 (s, C-11), 207,8 (s, C-8).

Aeruginolacton (7):
Bot vo dinh hinh,
[aj^-8,1"
(c 1,0,
CHCI3).
Negative-ion HR-FABMS: m/z 247,1324
[M-H]"
(C.jHigOj,
tfnh duac:
247,1334).
UV (MeOH):
k„,,
nm (log
f)
217 (4,05).
IR
(film):
v„,3,
3381, 1760 cm'.
CD (MeOH): Ae (nm): -0,79 (221), +0,17 (256) (c
6,4x10
'
M).
'H-NMR
(CDCI3):
8 1,46 m
(IH,
m, Ha-2), 1,60 (6H, s,
H-CHj,
IS-CHj)

1,80
(3H, s,
I3-CH3),
1,89
(IH,
m, H-3), 2,07
(IH,
m, Ha-6), 2,24
(IH,
m, H-3), 2,26
(IH,
m, Hb-2), 2,31
(IH,
m, Ha-9), 3,05
(IH,
m, Hb-9), 3,08
(IH,
m, Hb-6),
4,24
(IH,
brd,
7= 9,5 Hz, H-5), 4,77
(IH,
br s, H-l).
"C-NMR
(CDCI3):
8 8,6 (q, C-13), 16,8 (q, C-15), 17,3 (q, C-14), 25,3 (t, C-6)
25,6 (t, C-2), 38,4
(t,
C-3), 51,3 (t, C-9), 109,8 (d, C-8), 123,4 (d, C-5), 127,4 (s, C-

11),
131,7
(d,
C-l),
133,0
(s,
C-4),
134,2
(s, C-10), 162,4 (s, C-7), 171,9 (s, C-12),.
Guaianediol (8):
B6t
v6 dinh hinh,
[a]D-3,5°(c
1,82,
CHCI3).
UV (MeOH):
X„,,
nm (log
E)
219
(3,70).
'H-NMR
(CDCI3):
8 0,90 (3H, d, 7 = 7,2 Hz,
I3-CH3),
0,92 (3H, d, 7 = 6,4 Hz,
I2-CH3),
1,14 (3H, s,
I5-CH3),
1,20 (3H, s,

I4-CH3),
5,52
(IH,
br s, H-6).
"C-NMR
(CDCI3):
8 21,7 (q, C-14), 21,94 (q, C-13), 21,95 (q, C-12), 22,1 (t, C-
2),
23,1 (q, C-15), 25,6 (t, C-8), 37,8 (d, C-11), 41,0 (t, C-3), 43,2 (t, C-9), 50,9
(d, C-5), 51,3 (d, C-l), 75,8 (s, C-10), 80,8 (s, C-4), 121,9 (d, C-6), 150,1 (s, C-7).
Isoprocurcumenol (9):
Bot vo dinh hinh,
[a]"-62,5°
(c 1,82,
CHCl,).
UV (MeOH):
X^,^
nm (log
e)
248 (3,75).
CD (MeOH): Ae (nm): -1,15 (224), +0,94 (265), -0,51 (308)
(c
5,30x10"'
M).
'H-NMR
(CDCI3):
8 1,16 (3H, s,
I5-CH3),
1,36
(IH,

t, 7
=
15,6 Hz, H-5), 1,59
(2H, m, 2H-2), 1,75 (3H, s,
B-CH,),
1,80 (2H, m, 2H-6), 1,84 (3H, s, 12-
CH3),
1,94(1H,
t,7= 12,8 Hz, Ha-3), 2,76 (3H, br d, 7 = 14,4 Hz, Hb-3), 3,14
(IH,
d, 7
=
14,4 Hz, Ha-9), 3,24
(IH,
d, 7
=
14,4 Hz, Hb-9), 4,82
(IH,
s, Ha-
14),
4,84(1H, s,
Hb-14).
"C-NMR
(CDCI3):
8 22,7 (q, C-12), 23,7 (q, C-13), 25,1 (t, C-2), 25,6 (q,
C-15),
29,0 (t, C-3), 40,6 (t, C-6),
51,1
(d, C-l), 54,6 (t, C-9), 59,5 (d, C-5), 80,6 (s, C-4),
112,3

(t, C-14), 135,4 (s, C-7), 142,4 (s, C-10), 144,8 (s,
C-11),
204,2 (s, C-8).
Procurcumenol (10):
Bot vo dinh hinh,
[a]"+60,9° (r
0,86,
CHCI3).
UV (MeOH):
X„,^
nm (log e) 258 (4,30).
CD (MeOH): Ae (nm): -58,47 (214), -62,25 (237), +44,39 (278), +28,39 (334)
(c
7,69x10''M).
'H-NMR
(CDCI3):
8 1,15 (3H, s,
I5-CH3),
1,67 (3H, s, 12-CH3), 1,68 (3H,
s,
I3-CH3),
1,80 (3H, d, 7 = 1,2 Hz,
I4-CH3),
2,09
(IH,
dd, 7
=
16,0 Hz, 12,4
H/„
Hb-6),

2,53
(IH,
br d, 7 = 16,0 Hz, Ha-6), 5,77
(IH,
q, 7 = 1,2 Hz, H-9).
'^C-NMR
(CDCI3):
8 21,0 (q, C-12), 22,2 (q,
C-13),
23,1 (q,
C-15),
24,0 (q,
C-14),
26,7 (t, C-2), 28,4 (t, C-3), 39,6 (t, C-6), 50,2 (d, C-l), 53,6 (d, C-5), 79,8 (s, C-4),
128,8 (d, C-9), 136,2 (s, C-10), 136,3 (s, C-7), 155,4 (s,
C-11),
198,9 (s, C-8).
Aerugidiol (11):
B6t
v6 dinh hinh,
[a]"-17,0° (c
1,0, CHCI3).
UV (MeOH):
>.„„
(log e) 252 (3,84).
CD (MeOH): Ae (nm): -2,24 (213), +3,15 (246), -4,56 (283), +1,22 (315)
(c
6,6x10"'M).
'H-NMR
(CDCI3):

8 1,32 (3H, s,
I5-CH3),
1,70
(IH,
ddd, 7 = 13,6 Hz,
11,6
Hz,
6,4 Hz, Ha-2), 1,80 (3H, s,
U-CHj),
1,80
(IH,
m, Ha-6), 1,91 (3H, s,
I2-CH3),
1,91
(IH,
m, Ha-3), 1,93 (3H, s,
I3-CH3),
1,94
(IH,
m, H-5), 2,04
(IH,
dd, 7
=
11,6
Hz, 4,8 Hz, Hb-3), 2,13
(IH,
m, Hb-2), 2,56
(IH,
br d, 7 = 14,0 Hz, Hb-6),
4,18

(IH,
br s , OH), 4,38
(IH,
br s, OH), 5,71
(IH,
s, H-9).
"C-NMR
(CDCI3):
8 22,2 (q, C-14), 22,6 (q, C-13), 23,5 (q, C-12), 24,2 (q, C-15),
27,8 (t, C-6), 37,5 (t, C-3), 37,6 (t, C-2), 61,2 (d, C-5), 83,5 (s, C-4), 87,0 (s, C-l),
128,6 (d, C-9), 133,9 (s, C-7), 143,6 (s,
C-11),
151,3 (s, C-10), 195,5 (s, C-8).
Aeruginofuran (12):
Bot vo dinh hinh,
[a]"
-l,85°(c 0,21,
CHCI3).
Negative-ion HR-FABMS:
mJz
263,1283
[M-H]
(Tfnh duoc cho
C^.H^^O,:
263,1259).
UV (MeOH):
X^,^
nm (log e) 263 (3,43), 228 (3,36).
IR (film):
v^3,3400,

1707, 1651, 857 cm'.
'H-NMR
(CDCI3):
8 0,92 (3H, s,
I4-CH3),
1,38 (3H, s,
I5-CH3),
1,52
(IH,
m,
Ha-3),
1,69 (3H, m, 2H-2, Hb-3), 2,10 (3H, s,
I3-CH3),
2,47
(IH,
s, H-5), 2,62
(IH,
d, 7 = 16,8 Hz, Ha-9), 2,99
(IH,
d, 7 = 16,8 Hz, Hb-9), 3,57
(IH,
br s, H-
1),
7,0
(lH,s, H-12).
"C-NMR
(CDCI3):
8 8,8 (q, C-13), 14,4 (q, C-14), 24,3 (q, C-15), 27,8
(I,
C-2),

38,0 (t, C-3), 39,7 (t, C-9), 44,5 (s, C-10), 61,8 (d, C-5), 71,3 (s, C-4), 77,6 (d, C-
1),
119,1
(s,
C-11),
118,9 (s, C-7), 139,4 (d, C-12), 166,5 (s, C-8), 197,3 (s, C-6).
Zedoarofuran (13):
Bot vo dinh hinh, [a]"
+15,3°
(c 0,21,
CHCI3).
'H-NMR
(CDCI3):
8 1,11 (3H, s,
I4-CH3),
1,41 (3H, s,
I5-CH3),
2,72
(IH,
d, 7
= 17,4 Hz, Ha-9), 2,96
(IH,
d, 7 = 17,4 Hz, Hb-9), 2,15 (3H, s,
B-CH,),
2,44
(IH,
s, H-5), 3,57
(IH,
br d, 7 =
11,2

Hz, H-l), 7,05
(IH,
br s, H-12).
'^C-NMR
(CDCI3):
8 8,7 (q,
C-13),
12,7 (q, C-14), 26,7 (t, C-2),
31,2
(q,
C-15),
38,3 (t, C-3), 39,1 (t, C-9), 45,6 (s, C-10), 59,8 (d, C-5), 69,8 (s, C-4), 78,4 (d, C-
1),
118,7
(s, C-7), 120,4 (s,
C-11),
139,5 (d, C-12), 165,3 (s, C-8), 197,9 (s, C-6).
Aeruginon (14):
Bot vo dinh hinh,
[«]"
+173°
(c 0,76,
CHCI3).
'H-NMR
(CDCI3):
8 0,82 (3H, d, 7 = 6,4 Hz,
I2-CH3),
0,88 (3H, d, 7
=
6,4 Hz,

I3-CH3),
0,94 (3H, d, 7 = 6,8 Hz,
I4-CH3),
1,70 (3H, s,
I5-CH3),
2,78
(IH,
d, 7
= 10,8 Hz, Ha-9),
3,11
(IH,
d, 7 = 10,8 Hz, Hb-9), 4,38
(IH,
ddd, 7 = 10,4 Hz,
10,0 Hz, 6,0 Hz, H-2), 5,03
(IH,
dq, 7 = 10,0 Hz, 1,2 Hz, H-l).
10
*^C-NMR
(CDCI3):
8 16,9 (q, C-15), 18,5 (q, C-14), 19,7 (q, C-12), 21,0 (q, C-14),
29,9 (d, C-11), 42,5 (t, C-3), 44,2 (s, C-4), 44,3 (t, C-6), 54,1 (d, C-7), 56,2 (t, C-9),
68,2 (d, C-2), 129,6 (s, C-10), 133,8 (d, C-l), 210,3 (s, C-8), 215,0
(s,
C-5).
2.8 Ket qua va thao
lu^n
2.8.1 Phdn lap va xdc dinh cdu
true
cdc sesquiterpenoid

tiit
Nghe xanh (C. aff.
aeruginosa, Zingiberaceae)
Trong mot thdng bao trudc chung tdi da thdng bao ve ket qua xac dinh
mudi tam sesquiterpenoid
dS
bay hoi
tir than
re Nghe xanh (C. aff. aeruginosa)
sir
dung cac ky thuat GC va GC-MS, trong sd dd bay sesquiterpenoid da duoc
phan
lap
bang
each
ket hop ky thuat chung
cS^t
Kugelrohr va phan tach
sac
ky
cot tren silica gel [4]. Trong de tai nay mot phuong phap chiet va phan tach
mdi da duoc phat
tri^n
d^
nghien
ciru
cac hop
chS't
cd hoat tfnh sinh hoc
tir

than
re Nghe xanh (xem So do 2).
Bot khd than
rS
Nghe xanh duoc chiet bang MeOH va sau dd duoc phan
tach vdi cac dung moi
hOu
co cd do phan
cue
tang
dSn
de cho cac phan chiet
/7-hexan,
etyl axetat va
n-BuOH.
Bdt khd than re Nghe xanh
(C.
aff.
aeruginosa) duoc ngam chiet vdi MeOH d nhiet do phdng. Cac
ph^n
chiet
chiia cac hop chat trong than
rfi
Nghd xanh da duoc thu nhan bang
each
phan
bd
ph^n
chiet metanol thu duoc giua nudc va cac dung mdi cd do phan
cue

lang
dfo.
Bang phuong phap chiet chon loc nay cac
phSn
chiet
/i-hexan
(hieu sua't 3,14% so vdi luong nguyen lieu khd ban dau), etyl axetat (0,6%)
va
Az-butanol
(0,14%) da duoc
di^u
che.
Phan chiet
/i-hexan
duoc phan tach bang cac phuong phap
sac
ky cot
gradient tren silica gel,
Ircn
ODS va HPLC dieu che tren cot ODS
d^
cho
mudi bon
hgfp
chat tinh khiet, curdion (1), axit palmitic (2), zederon (3),
/^-sitosterol
(4), stigmasterol (5),
13-hydroxygermacrone
(6),
aeruginolacton (7), guaianediol (8), isoprocurcumenol (9), procurcumenol

(10),
aerugidiol (11), aeruginofuran (12), zedoarofuran (13) va aeruginon
(14).
Cac sesquiterpenoid 7, 12 la cac hcrp chat
hOfu
ca thien nhien mdi
va 14 la hop chat
I^n
ddu tien duac phan
l$p
tur thien nhien. Cau
true
hoa hoc cua cac hop cha't 1-14 da duoc xac dinh bang cac phuong phap
phd (UV, IR, MS,
ID
NMR va 2D NMR).
C^u
hinh
lap the cua cac hop
chat nay da duoc xac dinh bang phan tfch cac phd NOED, NOESY va CD.
11
So do 2: Chiet va phan tach phan chiet
/i-hexan tiir
than
r^
Nghe xanh
(Curcuma aff.
aeruginosa,
Zingiberaceae)
Curcuma

afT.
aeruginosa Roxb. (Zingibearceae)
ihAnrekh6(2kg)
i
n-hexan
Dg&m chie't
bing MeOH,
ohi^t
dd
ph^og
Ph^n chi^'l
MeOH (CA)
phAo b6
giira
?I20
va dung mdi
bihi
ca
EtOAc
n-BuOH
f
CAI
157
g
CA2
30
g
CA3
7g
Hi

g,
CC, silica
gel,
n-hexan,
n-hexau-HtOAc
(19:1,
9:1,
4:1,
2:1
va
1:1)
]
]
I
[
CAI-2
y,7g
CA3-7
45.4 g
CA8-9
11.2g
CAI
0-15
14.1
g
CAI
6-20
3.1
g
CA2I-25

4g
CA26-27
1.7
g
CA28-31
l>7g
5 g, CC, silica
gel,
/i-hexan-HiOAc(25:l)
lOg.CC,
sUicage!,
/j-hexau-KiC)Ac
(1
M.9:l)
1,2
3,4,5
6,7
8,9,10 11,12 13,14
dieu kien sic ky:
CC,
ODS gel.
MeOH-HjO
(3:2, 7:3} va
m.
prep. HPLC, ODS gel,
MeOH-HiO
(7:
i)
b.
peep.

HPLC, ODS gel,
MeOH-H^O
{i:2)
c prep. HPLC, ODS gel,
Me0H-H2O (1:1)
Phdn tich GC-MS cdc phdn doan chu'a cdc sesquiterpenoid de hay hd
Cac nhom phan doan 1 va 2 chua
chii
yeu cac hop cha't dl bay hoi da
diroc
phan tfch bang phuong phap GC-MS. Cac hop chat duoc xac dinh bang
each
so
sanh cac
ph6
khoi
lugng
cua chiing
v6i
thu vien
ph6
GC-MS Wiley Library va
vdri
ph6 khoi
lixong
cua cac
miu chu^n
cua chiing toi (Bang 1).
Ket qua cho tha'y cac hop chat chu yeu Uong nhom phan doan 1 la cac
sesquiterpen hydrocarbon (30,5%) vdi P-elemen (7,1%) va cac

sesquiterpenoid
chifa
oxy vdi furanodien (7,0%), curzerenon (12,8%), curcumol (5,6%),
germacron (11,2%) va curdion (6,9%) la cac thanh phan chinh. Trong nhom
phan doan 2 ham lugng cac sesquiterpenoid chira oxy da tang len nhieu lan so
voi trong nhom phan doan 1 (72,7% so vdi 43,5%), vci curzerenon (23,6%),
12
germacron (10,1%), curdion (29,4%) va curcumol (7,0%) la cac thanh phan
chfnh, trong khi do cac sesquiterpen hydrocarbon
chi
chiem 2,4%. Ngoai ra ca
hai nhom nay con chiia cac metyl este
ciia
cac axit beo o luong nho (tuong
iing
la3%val%).
Bang 1: Cac thanh phdn de bay hoi cua cac nhom phan doan 1 va 2
STT
1
2
3
4
5
6
7
8
9
10
11
12

13
14
15
16
17
18
19
Nhom
Thdi
gian Iim
(phut)
18,97
19,45
19,59
19,85
19,97
20,25
20,37
20,47
20,57
20,81
20,89
21,47
22,09
23,03
23,31
23,55
25,53
27,31
27,59

phan doan
1"
Ham
luong
7,1
2,2
0,9
1,6
4,0
1,7
2,7
3,7
7,0
0,6
1,6
4,4
12,8
5,6
11,2
6,9
1,2
1,4
0,4
Hgp cha't
P-Elemen
P-Cayophyllen
y-EIemen
Aromadendren
a-Humulen
y-MuuroIen

Germacren
D
P-Selinen
Furanodien
a-Muurolen
5-Cadinen
Germacren
B
Curzerenon
Curcumol
Germacron
Curdion
Metyl
hexadecanoat
Meiyl linoleai
Metyl
octadecanoat
STT
1
2
3
4
5
6
7
8
9
10
11
12

13
14
Nhom
Th5i
gian liru
(phut)
11,92
12,18
14,08
14,82
15,32
16,08
17,31
18,59
18,87
20,13
20,93
23,67
25,21
26,49
phan doan
2*"
Ham
luong
0,1
0,3
0,6
0,1
0,3
2,4

1,4
23,6
7,0
10,1
29,4
0,5
0,2
0,5
Ikyp chat
2-Oclenyl
axetat
2-Undecanon
p-Elemen
y-Elemen
a-HumuIen
Furanodien
Germacren R
Curzerenon
Curcumol
Germacron
Curdion
Metyl
hexadecanoat
Zederon
Metyl linoleai
•»
GC-MS:
60**C-220"C,
5**C/phut.
'»

GC-MS:
45'C-150"C,
lO'C/phiit,
dDcng
1
phiit,
150'C-250'C,
6'C/phut,
diing
15
phut.
*"*
% TIC duoc
do
irfin c6l
mao
qu^
HP-5.
13
Phdn lap vd xdc dinh cdu
triic
cdc sesquiterpenoid
Curdion (1):
Curdion (1) dugc phan lap sac ky vdi hieu suat 0,1 % so vdi lugng than
it
kho
tir
nhdm phan doan 2 chiia
chii
yeu la curzerenon (GC-MS: 23,6%) va curdion

(GC-MS:
29,4%). Hgp cha't nay nhan dugc dudi dang tinh the
hinh
kim, mau
trang, dnc
60-62*^C.
Ph6 IR cua 1
chi
ra su cd mat ciia nhdm carbonyl
(1696
cm')
va noi ddi (1665
cm"*).
Cong thtic phan
Ivr
cua 1 duoc xac dinh la
Ci5H-,402
bang
ph6 EI-MS (m/z 236,
M^).
Cac phuong phap ph6
ID
NMR
{'H-
va
''C-NMR)
va
2D NMR
('H-'H
COSY va HMQC) da dugc su dung trong viec xac dinh cau

Uuc
ciia 1. Ca'u
hinh
lap thd tuong doi cua curdion da
dirge
chiing tdi xac dinh bang
cac phan tfch cac ph6 NOED va NOESY. Ca'u
hinh
lap the tuyet ddi
ciia
curdion
dugc phan lap ttr C.
wenyujin
da dugc xac dinh bang
ph6 nhi6u
xa lia X
[121.
Sesquiterpenoid nay da dugc chirng to la cd hoat tfnh manh bao ve gan
1131.
Axit palmitic (2):
Axit palmitic (2) dugc phan lap
tir
nhdm phan doan 2 dudi dang tinh
the
hinh kim, khdng mau, dnc
52-54''C.
Tren ph6 IR xuat hicn dai hap thu
ciia
nhdm
hydroxy cua axit carboxylic (dai rong

tiT
3500-2500 cm'), nhdm carbonyl
ciia
axit (1703 cm"'). Hgp
ch^t
nay dugc xac dinh bang
each
so sanh phd EI-MS
(m/z 256,
C16H32O3,
M^)
vdi GC-MS Wiley Library.
Zederon (3):
Zederon (3) dugc phan lap sac ky
tir
nhdm phan doan 4 dudi dang tinh
the
hinh
kim, mau trang, dnc
150-152'^C,
vdi hieu
suS't
0,05% (so vdi lugng than
rC
khd).
Phd ER
chi
ra su cd mat ciia nhdm xeton a,P khdng no
(1698
cm') va

vong epoxit (1275, 1056 va 921
cm"*).
Cdng thiic phan tu ciia 3 dugc xac dinh la
14
Ci6H,803
bang phan tfch phd EI-MS (m/z 246,
M^).
Cac phan tfch phd 'H-,
"C-
NMR,
*H-*H
COSY va HMQC da cho cac
dfl
kien phd
phii
hgp vdi cau triic cua
zederon dugc phan lap tif C. zedoaria [14].
Ca^u
hinh tuong ddi cua zederon dugc
chiing tdi xac dinh bang phd NOESY. Ca'u triic lap
\hi
tuyet ddi cua hgp chat
nay da dugc xac dinh bang phd nhieu xa tia X [14].
P-Sitosteroi (4) va stigmasterol (5):
P-Sitosterol (4) va stigmasterol (5)
dirge
phan lap tir nhdm phan doan 4 dudi
dang mot hdn hgp kel linh hinh kim, mau trang, dnc
134-136*'C.
Phd

IR ciia
hdn hop
nay cho cac diinh ha'p thu
ciia
nhdm hydroxy (3434 cm') va ndi ddi (1648 cm"').
Phan tfch phd EI-MS cua hdn hgp cho phep du doan day la mot hdn hgp hai chat cd
cdng
Ihiic
phan tu la
C29H50O
(m/z 414,
M^,
4) va
C29H4«0
(m/z 412,
M^,
5). Cau
tnic ciia hai phytosterol nay dugc xac djnh bang cac dii kien phd 'H- va
'"^C-NMR
phii
hgp vdi cua cac cha't nay da dugc cdng bd' trong tai lieu tham khao |15|.
P-
Sitosterol dugc
sir
dung lam
nguyen
lieu dau de dieu che cac steroid hocmon
nhir
oesterol. Trong
Ihuc

te, p-sitosterol dugc diing dd didu Ui chiing
phinh
to tuyen tidn
li6t.
Cac steroid nay cung cd tac dung ddi vdi noc ran [16], lam giam
miic
cholesterol
va
lie
che
sU
phat tridn
ciia
ung thu ruot ket
[17].
13-Hydroxygermacron
(6):
Hgp cha't 6 dugc phan lap dudi dang bdt vd djnh hinh tir cac phan tach sac ky
nhidu
1^
nhdm phan doan 5, sir dung cac ky thuat sac ky vdi pha dao. Cac phan
tfch phd
*H-
va
*^C-NMR
da cho ihiy 6 la mot
dSn xuSft
cua germacron vdi
15
nhdm hydroxy gan vao vj

tri
cua nhdm metyl
C-13.
Cac du kien phd nay
phii
hgp vdi cac dQ
ki6n
phd
'H-
va
'^C-NMR
da dugc cdng bd trong tai lieu
1181
va
cho phep xac dinh ca'u
true
ciia 6 la
13-hydroxygermacron.
Ngoai tac dung
lic
che
sir
san sinh nito oxit (NO)
6
nong do
IC50
98
|iM
[19],
13-

hydroxygermacron (6) cdn dugc phat hien la cd hoat tfnh bao ve gan trong cac
thii nghidm
in vitro va in vivo nhu da dugc phat hien ddi vdi curdion (2) |20|.
Tuy nhien trong khi curdion dugc cho la thd hien tac dung qua co che bao ve lc
bao gan chdng lai ddc tfnh cua
D-GalN
(D-galactosamin),
thi co che tac dung
cua
13-hydroxygermacron
cd thd la bang
each tie
che
sir
san sinh
TNF-a
(tumor
necrosis
factor-a).
Aeruginolacton (7):
Hgp chat thien nhien mdi aeruginolacton (7) dugc phan lap dudi dang hot
25
vd dinh hinh vdi do quay
cue am
([a]^
-8,1°) tir nhdm phan doan 5 bang cac ky
thuat sac ky vdi pha dao. Cdng thiic phan tu cua 7 la
C,5H2o03
da dugc xac dinh
bang phd khdi lugng phan giai cao HR-FABMS ion

am
(m/z 247,1324,
[M-H|
).
Phd UV cho
dinh
ha'p thu
d
217 nm. Phd IR chi ra
sir
cd mat
ciia
cac nhdm chiic
hydroxy (3381
cm'')
va y-lacton (1760 cm"'). Cac
dir
kien phd
'H-
va
'^C-NMR
va cac tuong tac Uen phd HSQC cua 7 cho
th^fy
15 cacbon cua 7 dugc gan cho
ba nhdm metyl olefinic
[S^
1,60 (6H, s) va 1,80 (3H, s);
5^
tuong
iing

la 16,8
(q),
17,3 (q) va 8,6 (q)], mot nhdm xeton
[5^
171,9 (s)], mdt cacbon
xctal
|8(-
109,8 (s)], va cac cacbon cua ndi ddi
[6c
123,4 (d), 127,4 (s), 133,0
(s),
131,7
(d),
134,2 (s) va 162,4 (s), va bdn nhdm metylen
[8^
25,3 (t), 25,6 (t), 38,4
(t),
51,3
(t)].
Tren co sd cac phan tfch NMR ca'u
Uiic ciia
7 dugc gia thiet la mot
germacran lacton.
Su cd mat cua vdng y-lacton gdn vdi vdng mudi cacbon d C-7 va C-8 dugc
xac djnh bang cac tuong tac HMBC (Hinh 1) giua H-13
(^^
1,80) va C-7
|5,.
162,4 (s)],
C-U

[5c
127,4 (s)], va C-12
[5c
171,9 (s)J, va giira
H2-9
(S^
2,31 va
3,05) va C-8
[5c
109,8 (s)]. Hai ndi ddi the hai
l^n
dugc xac dinh la 0 cac vj tri
C-l(lO)
va C- 4, cdn cacbon
xetal
dugc gan d C-8 cung la ket qua cua cac phan
tfch cac tuong tac HMBC va su so sanh cac du kien phd
*^C-NMR
vdi cua
germacron [21].
16