-
B Y T
TRNG I HC DC H NI
NGUYN NGC T
NGHIN CU PHN LOI CC LOI
TRONG CHI GYMNEMA R.BR. THU
VIT NAM S DNG VN TAY HA
HC HPTLC
KHA LUN TT NGHIP DC S
H NI 2013
-
B Y T
TRNG I HC DC H NI
NGUYN NGC T
NGHIN CU PHN LOI CC LOI
TRONG CHI GYMNEMA R.BR. THU
VIT NAM S DNG VN TAY HA
HC HPTLC
KHA LUN TT NGHIP DC S
Ngi hng dn:
ThS. Phm H Thanh Tng
Ni thc hin:
B mn Thc vt Trng i
hc Dc H Ni
H NI 2013
-
Li cm n
Em xin gi li cm n chn thnh v su sc nht ti ThS Phm H Thanh
Tng. Thy tn tnh hng dn, gip v du dt em trong qu trnh lm
Nghin cu khoa hc v lm Kha lun tt nghip. Nh c s du dt ca Thy
m
em trng thnh ln tng ngy, hc c nhiu iu b ch v hon thnh c
kha lun tt nghip ny.
Em xin gi li cm n chn thnh ti PGS.TS. Trn Vn n v TS. Hong
Qunh Hoa. Thy c gip em rt nhiu trong qu trnh lm kha lun v
cho
em nhng li khuyn b ch trong hc tp v nghin cu.
Em xin gi li cm n chn thnh ti cc Thy, C trong b mn Thc vt.
Cc Thy C to iu kin tt nht em hon thnh Kha lun tt nghip ny
v Thy C lun l tm gng sng em noi theo v hc tp.
Em xin gi li cm n chn thnh n ch Chu Th Thoa v cc ch k thut
vin trong B mn Thc vt trng i hc Dc H Ni lun gip , to iu
kin thun li em hon thnh tt qu trnh lm thc nghim ti B mn.
Ti cng xin gi ti cc bn cng lm Nghin cu v Kha lun ti B mn
li cm n chn thnh v nhng gip chn tnh trong qu trnh lm thc
nghim.
Cui cng, con xin gi li cm n su sc ti ng b, b m v xin gi li
cm n ti em trai gip v ng vin tinh thn con trong sut nhng nm
thng hc i hc.
H Ni, thng 5 nm 2013
Nguyn Ngc T
-
MC LC
Danh mc t vit tt
Danh mc cc bng
Danh mc cc hnh
T VN 1
CHNG 1. TNG QUAN 2
1.1. TNG QUAN V CHI GYMNEMA R.BR. 2
1.1.1. c im thc vt 2
1.1.2. Thnh phn ha hc 3
1.1.3. Tc dng sinh hc 7
1.1.4. Mt s kt qu nghin cu tin hnh Vit nam 7
1.2. TNG QUAN V PHNG PHP PHN TCH HA HC
DC LIU
9
1.2.1. Phn tch ha hc bng phng php dung mi phn on 9
1.2.2. Phng php sc k lp mng (Thin layer chromatography) 9
1.2.3. Sc k lp mng hiu nng cao (High performance thin layer
chromatography)
16
CHNG 2. I TNG V PHNG PHP NGHIN CU 18
2.1 NGUYN VT LIU, THIT B 18
2.1.1. Nguyn vt liu 18
2.1.2. Thit b 19
2.2. NI DUNG NGHIN CU 20
2.2.1. nh tnh bt dc liu 2
2.2.2. Xy dng quy trnh phn tch HPTLC 20
2.2.3. Tin hnh phn tch ha hc mt s mu trong chi Gymnema
R.Br.
20
2.3. PHNG PHP NGHIN CU 20
2.3.1. nh tnh s b cc nhm cht trong cc loi nghin cu 20
-
2.3.2. Xy dng quy trnh phn tch nh tnh bng phng php sc
k lp mng
27
2.3.3. Tin hnh phn tch nh tnh cc phn on dch chit mt s
loi trong chi Gymnema R.Br.
28
CHNG 3. THC NGHIM, KT QU V BN LUN 30
3.1. NH TNH CC NHM CHT CHNH 30
3.2. QUY TRNH NH TNH BNG PHNG PHP SC K
LP MNG
31
3.2.1. Sng lc phng php chit xut 31
3.2.2. nh tnh cc phn on dch chit 32
3.2.3. Sng lc h dung mi pha ng 33
3.3. PHN TCH NH TNH CC DCH CHIT TON PHN
V PHN ON THEO QUY TRNH XuT
37
3.4. BN LUN 46
3.4.1. V phng php nghin cu 46
3.4.2. V nh tnh cc nhm cht chnh 46
3.4.3. V xy dng quy trnh nh tnh bng HPTLC 47
3.4.4. V phn tch nh tnh cc mu theo quy trnh xut 48
KT LUN 52
XUT 52
TI LIU THAM KHO 56
PH LC
-
DANH MC T VIT TT
CHDCND Cng ha dn ch nhn dn
DAGA Deacylgymnemic acid
HL Hi lu
HPTLC High performance thin layer chromatography (Sc k lp
mng hiu nng cao)
NK Ngm kit
NL Ngm lnh
STZ Streptozotocin
TLC Thin layer chromatography (Sc k lp mng)
UPGMA Unweighted Pair-Group Method with Arithmetical
Averages
UV Ultraviolet light (nh sng t ngoi)
VIS Visible spectrum (nh sng trng)
-
DANH MC CC BNG
Bng 1.1 Danh mc cc loi thuc chi Gymnema R.Br Vit
Nam theo Phm Hong H (2000) 2
Bng 1.2 Mt s tc dng sinh hc c cng b ca cc
loi trong chi Gymnema R.Br 7
Bng 1.3 Bng tm tt quy tc Stahl 11
Bng 1.4 Mt s h dung mi dng trong phn tch TLC 11
Bng 2.1 Danh mc mu c phn tch v m hiu 18
Bng 2.2 Tm tt cc phn ng nh tnh cc nhm cht trong
cc loi nghin cu 22
Bng 3.1 Kt qu cc phn ng nh tnh thnh phn ha hc
mt s loi trong chi Gymnema R.Br. 30
Bng 3.2 Kt qu khi lng cn thu c khi chit xut bng
cc phng php khc nhau 31
Bng 3.3 Tm tt kt qu nh tnh saponin v flavonoid trong
cc phn on dch chit 33
Bng 3.4 Gi tr Rf ca cc vch c trng chi Gymnema R.Br. 37
Bng 3.5 Bng h s tng ng cp i gia cc mu nghin
cu, s dng ch s Nei&Li 44
-
DANH MC CC HNH
Hnh 1.1 Cu trc ca cc acid gymnemic phn lp t
Gymnema sylvestre (Retz.) R. Br. ex Schult. 5
Hnh 1.2 Cu trc ca Gurmarin 6
Hnh 1.3 S tch chit phn on s dng dung mi khng
ng tan 10
Hnh 3.1 Sc k dch chit NL, NK, HL vi h dung mi
(S.2), hin mu di n UV 366nm 32
Hnh 3.2 Sc k mu DM vi h dung mi F.11 v S.2 hin
mu di n UV 366nm 35
Hnh 3.3 Quy trnh phn tch nh tnh cc phn on dch
chit bng HPTLC 36
Hnh 3.4 Sc k khai trin dch chit ton phn v phn
on ethylacetat vi h dung mi S.2 38
Hnh 3.5 Sc k khai trin dch chit ton phn v phn
on ethylacetat vi h dung mi F.11 39
Hnh 3.6 S khc bit gia cc mu ca loi Gymnema
sylvestre (Retz.) R.Br. ex Schult. 42
Hnh 3.7 S khc nhau gia cc mu ca loi Gymnema
inodorum (Lour.) Decnc. 43
Hnh 3.8 S khc nhau gia cc mu ca loi Gymnema
latifolium Wallich ex Wight. 43
Hnh 3.9 Cy phn loi da trn phn tch UPGMA h s
tng ng gia sc k ca cc mu 45
-
1
T VN
Trong nn y hc c truyn n Ayurveda, Dy tha canh (Gymnema
sylvestre (Retz.) R. Br. ex Schult.) c s dng t hn 2.000 nm nay
iu
tr i tho ng [41], [43]. Da trn kinh nghim ny, hng lot cc nghin
cu
c tin hnh chng minh tc dng sinh hc ca cy v xc nh hot cht
chnh trong cy, l acid gymnemic [36], [38] . Mt hng nghin cu mi
ang
c cc nh khoa hc quan tm p dng l tm kim v sng lc tc dng iu tr
i tho ng tng t Dy tha canh t cc loi khc trong cng chi
Gymnema
R.Br. Phng php ny da trn nguyn l l cc loi cng mt bc taxon thc
vt
thng c cc thnh phn ha hc tng t nhau do c xu hng c tc dng
sinh hc ging nhau [3]. Mt s nghin cu trn th gii chng minh c
tc
dng h ng huyt tng t trn cc loi Gymnema montanum Hook.f.
[15],
Gymnema inodorum (Lour.) Decnc [34], [33], Gymnema yunnanense
Tsiang [44]
Cho n nay, cc nghin cu trong nc c tp trung thc hin trn 2 loi
l
Gymnema sylvestre (Retz.) R.Br. ex Schult. [7] v Gymnema
latifolium Wallich ex
Wight. [3], [11]. Trong cc nghin cu ny, tc dng h ng huyt bc
u
c chng minh trn cc mu thu hi trong nc. Tuy nhin, cha c nghin
cu
no c thc hin mt cc h thng v s tng ng trong thnh phn ha hc
ca cc loi trong chi Gymnema R.Br..
Xut pht t thc t ny, ti Nghin cu phn loi cc loi trong chi
Gymnema R.Br. thu Vit Nam s dng vn tay ha hc HPTLC c thc hin
vi cc mc tiu sau:
- Xy dng quy trnh nh tnh cc thnh phn ha hc mt s loi thuc
chi Gymnema R.Br. bng phng php sc k lp mng.
- Xc nh s tng ng v thnh phn ha hc ca mt s loi trong chi
Gymnema R.Br. v xy dng cy phn loi thc vt chi da trn phn
tch nh tnh thnh phn ha hc.
-
2
CHNG 1. TNG QUAN
1.1.TNG QUAN V CHI GYMNEMA R.BR
1.1.1.c im thc vt
1.1.1.1.V tr phn loi
Theo h thng phn loi ca Takhtajan cng b nm 2009, Chi Gymnema
R.Br thuc H Thin l (Asclepiadaceae) c v tr phn loi nh sau
[39]:
+ Ngnh Ngc lan (Magnoliophyta)
+ Lp Ngc lan (Magnoliopsida)
+ Phn lp Bc h (Lamiidae)
+ B Long m (Gentianales)
+ H Trc o (Apocynacea)
+ Phn h Thin l (Asclepiadaceae)
+ Chi Gymnema
Theo Phm Hong H (2000), Vit Nam c 07 loi thuc Chi Gymnema
R.Br c th hin trong Bng 1.1. [8].
Bng 1.1. Danh mc cc loi thuc chi Gymnema Vit Nam theo Pham
Hong H (2000) [8]
STT Tn khoa hc Tn thng dng
1 Gymnema acuminatum (Roxb.) Wall. La ty nhn
2 Gymnema albiflorum Cost. La ty hoa trng
3 Gymnema inodora (Lour.) Decne. La ty khng mi
4 Gymnema latifolia Wall. ex Wight. La ty l rng, Dy tha canh
l
to
5 Gymnema sylvestre (Retz.) R. Br. ex
Schult.
Dy tha canh, dy mui, La
ty rng
6 Gymnema reticulatum (Moon) Alston
Cost.
Dy tha canh gn mng
7 Gymnema tingens (Roxb.) Spreng. Rau m, La ty nhum
-
3
Tuy nhin, theo ti liu c cng b mi nht v h Thin l Vit Nam
ca Trn Th Bch (2007), c 5 loi thuc chi Gymnema R.Br phn b Vit
Nam.
l: Gymnema inodorum (Lour.) Decne, Gymnema latifolium Wall. ex
Wight,
Gymnema yunnanense Tsiang, Gymnema reticulatum (Moon) Alst. v
Gymnema
sylvestre (Retz) R. Br. ex Schult. [2].
1.1.1.2.c im thc vt v phn b:
c im thc vt ca chi Gymnema R.Br: Cy leo, khng c r ph trn
thn. L mc i, khng nc. Cm hoa xim, dng tn n chm. Hoa nh. Thy
i
nh, hnh trng, u t, gc i c tuyn, t khi khng c tuyn. Trng hnh
bnh
xe; thy trng khng gp trong n, vn, ph nhau phi. Trng ph n, vy
trng
ph dnh trng, thng c cc hng lng xp dc theo trng. Ch nh dnh
nhau;
bao phn 2 , c phn ph nh, ht phn dnh thnh khi phn v c sp bao
bn
ngoi vch khi phn, khi phn khng c mm nh; c quan truyn phn c
gt
nh v 2 chui; khi phn hng ln; ch c mt khi phn trong mi phn. u
nhy phnh ln hnh trng, nh bu khng tht li thnh dng vi nhy. Ct nh
-
nhy hnh ng nhn u [4], [2], [8].
Phn b n , Nam Trung Quc, Vit Nam, Indonesia. Vit Nam, cc
loi thuc chi Gymnema R.Br phn b nhiu vng min trn c nc, t Hi
Dng, Hng Yn, Hi Phng, Bc Giang, Ninh Bnh n Thanh Ha, Ty
Nguyn, Ty Ninh [4].
1.1.2.Thnh phn ha hc
Cho n nay, cc nghin cu su v thnh phn ha hc c tp trung vo
loi Gymnema sylvestre (Retz.) R.Br. ex Schult. vi cc nhm cht
triterpene
saponin thuc 2 nhm oleane v dammarane. Saponin khung Oleane c cc
acid
gymnemic v gymnemasaponins, trong khi saponin khung dammarane l
cc
gymnemasides. Trong l cn xc nh c resine, albumin,
chlorophyll,
carbonhydrates, acid tartric, acid formic, acid butyric,
anthraquinon, alkaloid
inositole, acid hu c (5.5%), parabin, calci oxalate (7.3%),
lignin (4.8%), cellulose
(22%) [36], [35].
-
4
L Gymnema latifolium Wall. ex Wight bng cc phn ng nh tnh
thng
quy c xc nh l c cha cc nhm cht sau: saponin, tanin, flavonoid,
acid
amin, ng kh v coumarin [7].
Thnh phn tc dng chnh c xc nh l acid gymnemic, l tn chung
ca cc acid hu c thuc nhm saponin triterpenoid [23], [20], [36] .
Cc acid
gymmenic l cc dn cht th acyl (Tiglolyl, Methyltutylroyl,) ca
deacylgymnemic acid (DAGA). DAGA tng ng l dn xut th 3-O-
betaglucoronide ca gymemagenin [27]. Cu trc ca Gymnemagenin v 17
loi
acid gymnemic c minh ha trong Hnh 1.1 [36], [38].
Thnh phn th hai c quan tm nghin cu lin quan n c ch h
ng huyt l Gurmarin (Hnh 1.2), mt polypeptid c kh nng lm mt cm
gic
ngt m khng nh hng ti cc v gic khc, c phn lp t l Gymnema
sylvestre (Retz.) R.Br. ex Schult [25], [32].
-
5
Hnh 1.1a. Cu trc ca cc acid gymnemic phn lp t Gymnema
sylvestre
(Retz.) R. Br. ex Schult.
-
6
Hnh 1.2. Cu trc ca Gurmarin
Hnh 1.1b.Cu trc ca cc acid gymnemic phn lp t Gymnema sylvestre
(Retz.) R. Br. ex Schult. [37]
-
7
1.1.3.Tc dng sinh hc
Cho n nay, cc nghin cu v ha hc v tc dng sinh ch yu c thc
hin trn loi Gymnema sylvestre (Retz.) R.Br. ex Schult v c cng b.
Bn
cnh , mt s nghin cu cng c tin hnh trn cc loi khc nh Gymnema
inodorum (Lour.) Decne, Gymnema latifolium Wall. ex Wight v
Gymnema
yunnanense Tsiang (Bng 1.2)
Bng 1.2. Mt s tc dng sinh hc c cng b ca cc loi trong chi
Gymnema R.Br.
Loi Tc dng sinh hc
Gymnema sylvestre (Retz.) R.Br. ex
Schult
H ng huyt [16], [30], [26], [31].
Lm mt cm gic ngt [17].
H lipid mu v chng bo ph [41], [32],
[21], [25].
Khng khun [29].
Gymnema inodorum (Lour.) Decne c ch hp thu glucose [33],
[34].
Gymnema latifolium Wall. ex Wight H ng huyt [7].
Gymnema yunnanense Tsiang H ng huyt [44],
1.1.4. Mt s kt qu nghin cu tin hnh Vit Nam
Nm 2007, Anh V cng b mt s kt qu nghin cu sau [14]:
Khng nh tn khoa hc ca cy Dy tha canh l Gymnema sylvestre
(Retz.)
R. Br. ex Schult., H Thin l (Asclepidiaceae).
Khng nh cy Dy tha canh Vit nam cng c nhng tc dng h ng
huyt tng t nh nhng nghin cu trc c thc hin trn th
gii.
Nm 2008, Trng Th Tm cng b cc kt qu nghin cu sau [12]:
Xc nh trong l Dy tha canh c saponin, flavonoid, ng kh, acid
hu
c v acid amin.
-
8
Dch chit ton phn, dch chit nc v phn on dch chit ethylacetat
c
tc dng trn c m hnh chut bnh thng v m hnh chut gy tng
ng huyt bng STZ. Cc phn on dch chit n-hexan, chloroform
khng c tc dng trn c 2 m hnh th nghim. Phn on n-buthanol ch
c tc dng h glucose huyt trn m hnh chut gy tng ng huyt bng
STZ.
Nm 2009, Lng Thy An cng b cc kt qu nghin cu sau [1]:
Flavonoid tp trung ch yu trong phn on ethylacetat v
n-buthanol.
Saponin triterpenoid c trong 3 phn on: chloroform, ethylacetat v
n-
buthanol, trong tp trung ch yu trong phn on ethylacetat v n-
buthanol.
Phn lp c 1 flavonoid tinh khit k hiu l A1 t phn on
ethylacetat
d kin l Kaempferol.
Nm 2010, Nguyn Hng Giang cng b cc kt qu nghin cu sau [6]:
Xy dng v m t c quy trnh chit xut dng cao thuc tinh ch t dch
chit Dy tha canh (GS4) ti u vi cc iu kin: kch thc ca dc
liu, phng php chit xut, nng cn v pH trong phn ng acid ha.
La chn c 2 h dung mi nh tnh GS4 bng sc k lp mng:
H 1: Chloroform : Methanol : Acid formic (10 : 1.1 : 0,02) cho
10
vt vi thuc th hin mu l H2SO4 10%.
H 2: Chloroform : Aceton : Acid formic (3.3 : 1 : 0,02) cho 9 vt
vi
thuc th hin mu l H2SO4 10%.
phn lp c 2 cht tinh khit t GS4 k hiu l GS4A1 v GS5F2. D
kin GS4A1 l Myrtillogenic acid v GS5F2 l Glochioeriosides A.
Nm 2013, Phm H Thanh Tng cng b kt qu nghin cu [13]:
- M t c im hnh thi v cu to gii phu ca 26 mu trong chi
Gymnema R.Br. c thu thp t cc a phng khc nhau trong c nc;
Xc nh c h s tng ng c gi tr 0,44 0,88 s dng ch th
RAPD.
-
9
- Xc nh s c mt ca acid gymnemic trong dch chit methanol ca
08
mu ca 05 loi v gymnemagenin trong tt c dch chit thy phn.
- nh lng phn on giu saponin triterpenoid (GX4) trong 08 mu
trong
mu ca 2 loi cho t l cao nht l Gymnema latifolium Wallich ex
Wight thu Thi Nguyn (2,25%) v Gymnema yunnanense Tsiang thu
Kon Tum (2,57%). Thnh phn phn on GX4 c tnh tng ng cao khi
khai trin trn h dung mi chloroform : methanol (25:15) v so
snh.
1.2. TNG QUAN V PHNG PHP PHN TCH HA HC DC
LIU
1.2.1.Phn tch ha hc bng phng php dung mi phn on:
Phng php da trn phn cc v tan khc nhau ca cc cht trong
cc dung mi khc nhau. S tch chit phn on s dng dung mi khng ng
tan thng gp c th hin Hnh 1.3 [22].
1.2.2.Phng php sc k lp mng (Thin Layer Chromatography)
1.2.2.1. nh ngha
Sc k lp mng (TLC) l phng php phn tch trong dung dch cht
phn tch di chuyn trn mt lp mng cht hp ph mn, v c hay hu c,
theo
mt chiu nht nh. Trong qu trnh di chuyn, mi thnh phn chuyn dch vi
tc
khc nhau, ty theo bn cht ca chng v cui cng dng li nhng v tr
khc
nhau [22], [10], [5].
a. Cht hp ph:
Hin tng hp ph l s tch t mt cht th kh hoc trong dung dch trn
b mt mt cht rn. Cc cht hp ph c dng trong sc k lp mng hu ht l
cc oxid khng tan, cc oxid hydrat ha v cc mui. Cc cht hp ph thng
dng
l silica, alumina, octadecasilica, cellulose, dextran gel,
polyamid, hoc cc resin
trao i ion khc. Silicagel l cht hp ph thng gp nht. Kh nng hp ph
ca
silicagel l do cc nhm silanol c nhm hydroxyl. Khi bo qun trong
iu kin
thng, silicagel d dng hp ph nc trong khng kh, lm gim kh nng
hp
ph ca n. Do vy, silicagel thng c sy 100-120oC loi nc hp ph
-
10
m khng lm mt i nhm hydroxyl ca n v silanol. Kh nng hp ph ca
silicagel cao nht khi c hot ha 105oC trong 2 gi [10].
Hnh 1.3. S tch chit phn on s dng dung mi khng ng tan [22]
b. Dung mi:
Tc di chuyn ca mt cht trn b mt cht hp ph, ph thuc vo
dung mi. Tiu chun quan trng nht la chn mt dung mi chnh l phn
cc ca dung mi v pH ca dung mi. Chn la dung mi cho sc k, cch tt
nht
Dc liu
Dch chit methanol
Phn on n-hexan
Lp nc
Phn on
EtOAc Lp nc
Phn on
n-butanol
Phn on nc
Chit vi n-butanol bo ha nc
Bay hi ht MeOH, lc vi ng lng ethylacetat
C c, ha tan vo methanol 90%
Lc vi n-hexan
Chit bng methanol
-
11
l bng thc nghim. nh hng cho thc nghim ny, c th p dng quy tc
tam gic ca Stahl (1960s) (Bng 1.3).
Bng 1.3. Bng tm tt quy tc Stahl [10]
Mu th Cht hp ph H dung mi khai trin
Phn cc km
(phytosterol, coumarin,
terpenoid, carotenoid, ...)
Phn cc mnh
(silicagel, nhm oxid)
Phn cc km
(hexan, toluen, benzen...)
Phn cc mnh
(glycosid, polyphenol...)
Phn cc km
(silicagel RP...)
Phn cc mnh
(AcCN, methanol, nc...)
Mt khc, pH ca dung mi cng ng vai tr quan trng. Khi sc k cc
polyphenol c tnh acid, nu cho thm acid (acid formic, acid acetic
bng) vo dung
mi s lm gim tng tc gia polyphenol v silanol, mu s di chuyn nhanh
hn
v vt gn hn. Ngc li, khi sc k cc alcaloid base c tnh kim, ngi
ta
thng thm mt t kim (amoniac, triethylamin, diethylamin,
dimethyl,
fornamid...) vo dung mi khai trin. Cc cht ny c gi l cht iu chnh
[22],
[10], .
Bng 1.4. Mt s h dung mi dng trong phn tch TLC [22]
H dung mi Cht hp ph ng dng
Hexan:ethyl acetat Silica gel Tch cht t phn cc hoc phn
cc trung bnh
Petroleum ether :
Chloroform
Silica gel Tch cht t phn cc, v d nh
cc dn xut acid cinamic,
coumarins
Chloroform:aceton Silica gel Tch cht phn cc trung bnh
Benzen:aceton Silica gel Tch hp cht thm
Chloroform:methanol Silica gel Tch cht phn cc, v d nh
glycoside v polyphenol
Butanol:acid acetic:nc Silica gel Tch glycosid v ng
-
12
Methanol:acetonitril:nc C18/C8 TLC pha o
i vi cc acid hoc base, thm
lng nh acid hoc base c th
ci thin sc k
Methanol:ethanol:nc Polyamide Ph bin cho polyamide
Methanol:ethanol:nc Cellulose Tch cht phn cc nhiu nh
ng hoc glycosid.
1.2.2.2. ngha - ng dng
Sc k lp mng l mt phng php n gin, kinh t v nhanh gn c th
c dng cho phn tch nh tnh v nh lng cng nh l tinh ch hp cht t
thin nhin. Thc t, phng php ny rt hu ch trong nhn dng mt hp
cht
bit v pht hin s c mt ca nhiu loi hp cht khc trong cy c, v d
nh
phenols, steroids, alkaloids, flavonoid, v coumarin. TLC cn ng
vai tr nh mt
cng c b tr ti u ha iu kin ca sc k ct, bao gm la chn pha ng v
pha rn, xc nh cht phn lp t sc k ct, cng nh l xc nhn s tinh khit
ca
phn on phn lp c [22].
1.2.2.3. Phng php tin hnh
a. Chun b bn mng:
Bn mng nn c ra sch trc khi tin hnh khai trin do lp hp ph
silicagel c th d dng b nhin bn bi thi gian v iu kin bo qun km.
Bn
mng c nhng vo mt b m. Dch ra c th l methanol, methanol
dichloromethan (1:1) hoc isopropanol, c chy qua ht bn mng. Bn
mng
ra, sau c hot ha li trong t sy [18].
b. Chm mu:
Chm mu bng phng php th cng, bn t ng, hay t ng u c th
c dng. V mt c bn, dch th v dch chun c chm bng ng mao qun.
Tuy nhin, nn s dng phng php bn t ng hoc t ng phun mu.
Khong cch t v tr chm mu n ra di ca bn mng ph thuc vo kch
-
13
thc ca bn mng. Nhn chung, khong cch nn l 10-15mm i vi bn tin
dng (conventional plate) v 8-10mm i vi bn hiu nng cao (high
performance
plate). Phn ln s dng bn c kch thc 10mm. Ngy nay, ngi ta thng
chm
theo bng, di bng khong t 8-10mm i vi bn tin dng v 5-8mm i vi
bn hiu nng cao. Khong cch gia cc im/bng thng l 5, 8, 10mm,
ty
thuc vo iu kin lm vic thc t. c c kt qu tt nht, bng nn mng
nht c th, thng nh hn 1mm. Ch khng lm hng b mt bn mng trong
lc phun mu [18].
c. Khai trin:
Bn mng sau khi c phun mu c t vo bnh khai trin bo ha
dung mi khai trin trong 15 pht. Th tch pha ng khong 8 -10ml i vi
bn
10cm x 10cm. Thng thng, khong khai trin nn l 70-90mm i vi bn
tin
dng v khong khai trin hp l v l tng nht trn bn hiu nng cao l
50-
70mm [18].
d. Hin mu:
Nu cc vt chm sc k c mu th hnh nh c th c ghi li di nh
sng trng. Nu vt chm sc k c hunh quang UV th quan st di nh n
t
ngoi l cn thit. i vi mt s cht khc, cn phun hoc nhng vo thuc
th
c trng hin mu v quan st trc tip hoc sau khi sy. Phng php
nhng
c u im l to mt mu ng nht cho c bn mng nhng li d ha tan cht
phn tch vo dung dch thuc th hoc lm cho vt b ko ui. Vic gi n
nh
thi gian v nhit lm nng c bit quan trng. Tro ha bn mng d dng
xy
ra nu nhit qu cao hoc thi gian qu lu. Nu acid sulfuric c dng
lm
thuc th hin mu th bn mng rt d b tro ha. Mt s cht b bin mu
theo
thi gian, c bit l sau khi phun thuc th [18].
1.2.2.4. Cc yu t chnh nh hng n qu trnh sc k
a. X l mu v chun b dung dch th
Thng thng, dc liu thng cha nhiu hp phn phc tp ca cc cht
c bit n hoc cha c bit n nn sc k bn mng th hin c cng
-
14
nhiu thng tin ha hc cng tt. Tuy nhin, c th c mt bn sc k r rng
v
sch s vi phn gii cao cc cht mc tiu th mu nn c tinh khit ha v
x l trc khi tin hnh TLC. Dung mi l tng s dng trong chun b
dung
dch th nn c ha tan chn lc i vi cht phn lp v nht thp hn cng
nh l im si thp hn. Trong trng hp ny, methanol v ethanol thng
c
coi l mt dung mi ti u cho chit mu trong TLC. Sau , tinh ch hoc
ra dch
chit ton phn thng c tin hnh tch hp cht mong mun ra khi nhiu
tp cht khng mong mun [18].
b. K thut chm mu
y l mt bc quan trng trong phn tch sc k lp mng c c bn
sc k c phn gii cao. chnh xc ca cc phn tch nh lng cng b nh
hng nhiu bi bc ny v chm mu khng t yu cu chnh l mt trong
nhng nguyn nhn gy sai s. Trong k thut chm mu, cn ch mt s im
nh sau:
Lng mu chm: Kh nng cha mu ca bn mng l c gii hn. Nu
lng mu vt qu kh nng ny th kh nng tch s gim ng k. c bit l
cc bn mng thng mi hin nay, c trng sn bi cc tiu phn silicagel
mn
(11-12m v 5-7m), ci thin c kh nng tch nhng li gii hn kh nng
cha mu. Do , cn thm d lng mu chm hp l c c phn gii v
lp li cao nht [18].
ht m ca dung mi dch chit s c th lm thay i kh nng khai
trin. Nu dung mi dch chit thn nc, th sau khi chm, im chm s hp
ph
m t khng kh c bit l trong iu kin kh hu m t v nh hng ln ti
phn tch sc k lp mng. Do , sau khi chm cn sy kh loi b m ngay t
u
nhng trnh nhit qu cao lm bin i cht phn tch.
Hng c hc trn b mt bn mng do thit b chm mu cng l mt trong
nhng nguyn nhn gy nn kt qu sc k khng tt trong phn tch nh
lng.
Tuy nhin, cc bn mng thng mi c chc cao v thit b phun mu bn t
-
15
ng hay t ng c th hon ton loi b kh nng lm hng b mt bn mng
[18].
c. nh hng ca s hp ph v m tng i n sc k
Hot ng ca cht hp ph c quyt nh bi nng lng b mt v din
tch b mt ca n. Nng lng b mt cng ln, din tch b mt cng rng th
kh
nng hp ph cng cao. Kh nng hp ph silicagel l do nhiu nhm silanol
trn
b mt ca n, nhng nhm hydroxyl hot ha v hydroxyl t do c th to lin
kt
hydro vi cc cht phn cc hoc cha bo ha. S lng nhm hydroxyl hot
ng
v hydroxyl t do s quyt nh hot ng ca silicagel. Silanol l nhm thn
nc
v n c th d dng lin kt vi nc to thnh nhm silanol hydrat ha v
mt
kh nng hp ph. Do , bn mng cn c hot ha nhit cao chuyn
ht cc nhm silanol hydrat ha thnh silanol t do, ti u ha kh nng hp
ph
ca bn mng. y l qu trnh thun nghch. Nhit hot ha thng l 105-
1100C, v bn mng sau khi hot ha phi trnh tip xc vi mi trng th
nghim
trc khi tin hnh chm mu.
m nh hng ln ti qu trnh sc k. Mt vi thnh phn v h dung
mi pha ng s dng c th c nh hng ti m tng i. iu kin m
30-70% s m bo s n nh ca qu trnh chy sc k. c c lp li cao
gia cc phng th nghim khc nhau, trong cc ma khc nhau th nn kim
sot
m trong qu trnh th nghim [18].
d. Vai tr ca bay hi dung mi:
Khng ging nh sc k ct, trong sc k bn mng tn ti mt pha hi hay
cn gi l pha th ba (third phase). Trong khong khng gian ca bung
khai
trin, pha hi ng vai tr quan trng v tham gia vo qu trnh khai
trin. Do
dung mi pha ng cn c bo ha trong khong thi gian nht nh, thng
thng l 20 pht, m bo qu trnh khai trin t kt qu ti u [18].
e. nh hng ca nhit trong phn tch sc k lp mng:
Trong mt iu kin m tng i nht nh, Rf c xu hng cao hn khi
khai trin mu trong nhit cao. Tuy nhin, dao ng gi tr Rf ni chung
khng
-
16
vt qu 0.02 nu thay i trong nhit khai trin ri vo trong khong
50C.
Nh vy, nh hng ca nhit trong sc k lp mng khng qu ln. Tuy vy,
nu s dao ng nhit khai trin t ti mc cao th kt qu sc k bn mng
s
b nh hng ln. Th nht, im si, p sut hi v khi lng ring ca mi
dung
mi trong pha ng thay i dn n thay i trong t l mi phn trong dung
mi
khai trin. Th hai, nhit thay i chc chn s thay i phn nc trong
pha
ng hai pha dn n thay i phn cc ca pha ng v thay i phn tch sc
k [18].
f. Vt liu lm bn mng v vt liu trng bn mng.
1.2.3. Sc k lp mng hiu nng cao (High performance thin layer
chromatography HPTLC)
Sc k lp mng hiu nng cao (HPTLC) l mt hnh thc tn tin nht ca
cng c TLC nhng n khng n gin m ch TLC cng c tin hnh trn bn
mng c hiu sut c bit cao. HPTLC l mt khi nim ln bao gm phng
php lun c tiu chun ha da trn c s khoa hc v nhng phng php nh
tnh, nh lng chnh thng. Dng c tinh vi c iu khin bi mt phn mm
tch hp m bo tnh ng dng, tin cy v lp li cao nht ca cc s liu
a ra. Trong phn tch HPTLC, cc thng s ca qu trnh phn tch c ghi
li
v c kim sot cht ch, do , m bo c lp li cao. ng thi, cc
bc ca qu trnh phn tch bao gm phun mu, khai trin mu v nhn din
vt
c tin hnh bn t ng, gim thiu ti a cc sai s c th gp phi. Qu
trnh
phun mu c tin hnh bn t ng, m bo chnh xc th tch mu phun, tc
phun v khng c nguy c hng c hc bn mng. Trong qu trnh khai trin,
iu
kin khai trin v nhit v m c kim sot cht ch v ghi li y , m
bo lp li ca kt qu khi tin hnh nhng phng th nghim khc nhau. H
thng n UV tch hp my nh v h thng phn mm gip phn tch s liu ng
dng trong nh tnh v nh lng.
Ngy nay, khi phn tch TLC c tiu chun ha, ng dng ca phng
php ny ngy cng ln. Nhiu nghin cu s dng HPTLC trong xy dng
-
17
fingerprint xc nh cc marker ha hc c trng ca cc loi thc vt cng
nh
phn loi cc loi v di loi v xy dng thnh b c s d liu tra cu
[42].
-
18
CHNG 2. I TNG V PHNG PHP NGHIN
CU
2.1. NGUYN VT LIU, THIT B
2.1.1. Nguyn vt liu
Mu l ca loi thuc chi Gymnema R.Br c trng v thu hi nhng
vng khc nhau trong phm vi lnh th Vit Nam. Ring mu c pht hin v
thu
hi vng Bolikhamxay Cng ha dn ch nhn dn Lo (CHDCND Lo) cng
c a vo nghin cu do chng phn b gn bin gii Vit Lo, c iu kin kh
hu tng t vng Trung B v Ty Nguyn Vit Nam. Tn, a im, thi gian
thu
hi c th hin trong Bng 2.1.
Bng 2.1. Danh mc mu c phn tch v m hiu
Mc ch
nghin cu
Tn loi a im thu hi Thi
im thu
hi
M
hiu
mu
nh tnh
cc nhm
cht chnh
trong cc
loi
Gymnema latifolium
Wall. ex Wight
Thi Nguyn 3/2012 GL
Gymnema sylvestre
(Retz.) R.Br. ex Schult.
Thanh Ha 4/2013 GS
Gymnema yunnanense
Tsiang
k Lk 4/2013 GY
Gymnema inodorum
(Lour.) Decne
H Ni 5/2013 GI
Gymnema sp. CHDCND Lo 2/2011 GS1
Sng lc
phng
php chit
xut
Gymnema latifolium
Wall. ex Wight
Thi Nguyn 3/2012 CX1
CX2
CX3
Sng lc h Gymnema latifolium Thi Nguyn 3/2012 DM
-
19
dung mi Wall. ex Wight
Phn tch
nh tnh
bng
HPTLC
Gymnema latifolium
Wall. ex Wight
Thi Nguyn 3/2012 GL1
Ty Ninh 3/2013 GL2
Gia Lai 3/2013 GL3
Ha Bnh 5/2013 GL4
Gymnema sp. CHDCND Lo 2/2011 GS1
Gymnema sylvestre
(Retz.) R.Br. ex Schult.
Thanh Ha 4/2013 GS2
Thi Nguyn
(Hoa trng)
5/2013 GS3
Thi Nguyn
(Hoa vng)
GS4
Thi Nguyn
(Hoa )
GS5
Gymnema yunnanense
Tsiang
c Lk 4/2013 GY1
Gymnema inodorum
(Lour.) Decne
Dng Ni, H Ni 5/2013 GI1
Vn thc vt, H
Ni
GI2
L ca cc mu c sy kh, xay c th (2000/355), chit xut theo quy
trnh xut v c dng chit phn on v chm sc k HPTLC.
2.1.2. Thit b
T sy SHELLAB.
My xay dc liu.
B chit hi lu 1000ml.
Phu chit 500ml.
My ct quay thu hi dung mi Buchi.
B iu nhit Memmert.
-
20
Cn k thut Sartorius.
My li tm Hettich Zentrifugen.
H thng sc k bn mng hiu nng cao HPTLC CAMAG (h thng bao
gm: thit b chm mu bn t ng Linomat 5, thit b pht trin t ng
ADC2, bung chp nh TLC visualiser, c iu khin thng qua phn
mm winCAT v phn mm phn tch VideoScan trn my tnh).
Cc c m, a thy tinh, ng nghim, lam knh, v.v
2.2. NI DUNG NGHIN CU
2.2.1. nh tnh bt dc liu
nh tnh cc nhm cht chnh trong bt dc liu ca tt c cc mu bng
cc phn ng ha hc c trng.
2.2.2. Xy dng quy trnh phn tch HPTLC
- Tin hnh nghin cu s nh hng ca cc phng php chit xut n
hm lng cn ton phn v sc k ca dch chit tng hp.
- Theo cc ti liu thu thp c, thnh phn ch yu trong l ca GS ch
yu l saponin v flavonoid, do , phn tch ch yu hai nhm cht ny.
- Sng lc h dung mi chy sc k lp mng hiu nng cao cho nhm cht
saponin v flavonoid.
- Xy dng v m t quy trnh phn tch ti u t cc yu t nghin cu.
2.2.3. Tin hnh phn tch ha hc mt s mu trong chi Gymnema R.
Br.
- Chit xut v tch phn on theo quy trnh m t t kt qu sng lc.
- Chy sc k HPTLC trn h thng sc k bn mng hiu nng cao HPTLC
ca CAMAG vi iu kin tin nh trong quy trnh phn tch xy dng
mc trn.
2.3. PHNG PHP NGHIN CU
2.3.1. nh tnh s b cc nhm cht trong cc loi nghin cu
Xc nh cc nhm cht trong cc loi: Gymnema sylvestre (Retz) R.
Br.,
Gymnema inodorum (Lour.) Decne, Gymnema latifolium Wall. ex
Wight v
Gymnema yunnanense Tsiang bng cc phn ng ha hc nh tnh thng
quy.
-
21
Trc khi tin hnh cc phn ng ha hc, cc dch chit c chun b nh
sau [9]:
Dch chit ether: Chit 10-25g bt dc liu bng diethyl ether bng
cch
lc trong mt bnh nn trong vng 10-20 pht cho ti khi dch chit ether
khi bc
hi khng cn li lp cn m trn mt knh ng h. Gp dch lc c dch
chit ether.
Dch chit cn: Chit tip b dc liu ( c x l bng n-hexan loi
chlorophyll) bng cn cao (hoc methanol) trong bnh nn vi sinh hn
hi lu
20-30 pht trn bp cch thy, thc hin 2-3 ln. Gp dch chit, lc v c li
(nu
cn) thu c dch chit cn.
Dch chit cn thy phn: Mt phn dch chit cn c thy phn nh
tnh cc aglycon sau khi thy phn. Thm 10ml acid hydrochloric 10%
vo 15ml
dch chit cn v un hi lu trn bp cch thy trong 30 pht, thm 10ml
nc.
ngui, cho hn hp vo bnh gn v chit bng ether ethylic ( 15ml x 3
ln). Dch
ehter c dng nh tnh cc aglycon.
Dch chit nc: B dc liu l tip tc c chit nng vi nc trong
bnh nn trn bp cch thy si. Gp cc dch chit, ngui, lc v c li
(nu
cn) thu c dch chit nc.
Dch chit nc thy phn: Mt phn dch chit nc c thy phn
nh tnh cc aglycon sau khi thy phn. 15ml dch chit nc c thy phn
bng
10ml acid hydrochloric 10% v un hi lu trn bp cch thy 30 pht.
ngui,
chit hn hp bng ether ethylic (15ml x 3 ln). Dch ether c dng nh
tnh
cc aglycon.
Cc dch chit chun b c dng tin hnh cc phn ng nh tnh
c tm tt trong Bng 2.2.
Bng 2.2. Tm tt cc phn ng nh tnh cc nhm cht trong cc loi
nghin cu
Nhm cht Phn ng M t Dng tnh
Glycosid tim Phn ng Ly 5ml dch cn cho Xut hin mu tm
-
22
Nhm cht Phn ng M t Dng tnh
Raymond
Marthoud
nh tnh vng
lacton 5 cnh
vo chn s bc hi ti
cn. Ha tan li cn vi
2ml cn, gn dch cn
vo ng nghim nh.
Thm 2-3 git dung dch
m-dinitrobenzen 1%
trong cn 96 (ri thm 3
git KOH 5%).
nh tnh ng 2-
desoxy
Ly 5 ml dch cn cho
vo chn s bc hi n
cn. Ha tan cn vi 5ml
thuc th xanthydrol
khuy cho tan ht cn.
y ng nghim v un
cch thy trong 5 pht.
Xut hin mu
hng n mn
Saponin
Phn ng
Salkowski
Nh tng git dung dch
H2SO4 c vo 1ml dch
chit chloroform.
Mu thay i t
vng, , l xanh
l hay l tm
Phn ng to bt
Ly 5ml dch chit cn,
c trn bp cch thy ti
cn, ha tan li bng 5ml
cn 25% trn bp, lc
vo ng nghim. Nh 2
3 git dch lc vo 2 ng
sau:
- ng 1cha 5ml
HCl 0,1N.
- ng 2cha 5ml
Nu ct bt 2
ng cao bng nhau
v bn nh nhau
th c Saponin
triterpenoid.
Nu ct bt ng
kim cao hn th
c Saponin
steroid.
-
23
Nhm cht Phn ng M t Dng tnh
NaOH 0,1N.
Sau , bt ming v lc
mnh 2 ng trong 15
giy.
Phn ng
Rosenthaler
Nh vi git vanilin 1%
vo ng nghim cha
1ml dch chit, sau h
nng ng nghim trn
ngn la n cn.
Xut hin mu hoa
c
Flavonoid
Phn ng Cyanidin
nh tnh cc
flavonoid c nhn
-pyron v -
dihydropyron
Ly 5ml dch chit cn,
bc hi n cn khong
2ml. Thm mt t bt Mg
vo ng nghim. Sau
nh 3 5 git acid
hydrochlorid c vo ri
yn vi pht.
Xut hin mu
cam, thm,
ti.
nh tnh
anthocyanosid
Thm 2-3 git dung dch
acid hydrochloric 10%.
Kim ha bng dung dch
natri hydroxid 10%.
Dung dch cho
mu hng ti
, chuyn sang
xanh khi kim
ha.
nh tnh
proanthocyanidin
Thm 2ml dung dch acid
hydrochloric 10% v un
trong bp cch thy 10
pht.
Dung dch c mu
hng ti .
Alcaloid Ly khong 5ml dch chit cn, bc hi ti cn. Ha tan cn
trong
2-4ml dung dch acid hydrochloric 5%. Chia dung dch acid vo 4
-
24
Nhm cht Phn ng M t Dng tnh
ng nghim v nh tnh bng cc thuc th sau.
Thuc th
Dragendorff
Ha tan 8g Bitmut nitrat
kim trong 20ml dd
HNO3. Ha tan 27,2 g KI
trong 50ml nc. Trn
hai dung dch li ri thm
nc cho 100ml.
Ta vng cam n
Thuc th Mayer
Ha tan 1,36g HgCl2
trong 60ml nc. Ha tan
5g KI trong 10ml nc.
Trn 2 dung dch li ri
thm nc cho 100ml.
Ta trng hay
vng nht
Thuc th Wagner
Ha tan 1,27g Iod v 2g
KI trong 20ml
nc.Thm nc cho
100ml
Ta nu
Anthraquinon Phn ng
Borntraeger
nh tnh dng t do:
thm 1ml dung dch NH3
10% vo ng nghim 1
ml dch chit ether hoc
chloroform ri lc nh.
nh tnh dng ton
phn: thm 1ml dung
dch NH3vo ng nghim
cha 1ml dch chit
chloroform ri lc nh.
Tip tc nh tng git
dung dch NaOH 10% v
Xut hin mu
sim
-
25
Nhm cht Phn ng M t Dng tnh
lc nh.
Coumarin
Phn ng m ng
vng lacton
Cho 1ml dch chit cn
vo 2 ng nghim sau:
- ng 1: cha 0.5ml
dung dch KOH
10%
- ng 2: cha 0.5ml
nc ct
un cch thy c 2 ng
trong 2 pht. ngui
ri quan st. Thm vo c
2 ng nghim mi ng
2ml nc ct. Lc u ri
quan st.
Acid ha ng 1 bng vi
git HCl c.
Ban u, ng 1 c
mu vng hoc ta
c mu vng,
ng 2 trong.
Sau khi thm nc
ct, ng 1 trong
sut, ng 2 c ta
c.
Sau khi acid ha,
ng 1 s tr li ta
c nh ng 2.
Phn ng Diazo
Thm 2ml dung dch
NaOH 10% vo ng
nghim cha 1ml dch
chit ri lc k, sau
thm vi git thuc th
Diazo mi pha.
Xut hin ta
gch.
Tanin
Phn ng vi
FeCl3
Ly 2ml dch chit nc
vo ng nghim ri thm
2 git dung dch FeCl3 5
%.
Xut hin mu
hoc ta mu xanh
en hoc xanh ru.
Phn ng vi ch
acetat
Ly 2ml dch chit nc,
thm 2 git ch acetat
Xut hin kt ta
bng
-
26
Nhm cht Phn ng M t Dng tnh
10%.
Phn ng vi dung
dch gelatin
Ly 2ml dch chit nc,
thm 5 git dung dch
gelatin 1%.
Xut hin kt ta
bng trng
Tinh du Cn c mi thm
Ly khong 5ml dch
ether cho vo chn s,
bc hi ti cn. Nu cn
c mi thm nh, tip tc
thm cn cao ri li
bc hi n cn.
Cn c mi thm
nh c trng.
ng kh Phn ng vi
thuc th Fehling
Ly 1ml dch chit nc
vo ng nghim ri thm
vi git thuc th
Fehling, sau un si
trong 2 pht.
Xut hin ta mu
nu
Acid hu c Phn ng vi
Na2CO3
Thm vo dch chit
nc mt lng tinh th
Natri carbonat.
Xut hin bt kh
Acid amin Phn ng vi
ninhydrin
Cho 10ml nc ct vo
ng nghim cha 1g bt
dc liu, un trong 5
pht ri lc nng. Ly
2ml dch lc, nh 2 3
git thuc th ninhydrin
3%, ri un cch thy si
trong 10 pht
Xut hin mu
xanh tm
Sterol Phn ng
Libermann
Ly 1ml dch chit ether
du ha, bc hi dung
Mt phn cch
gia hai lp c
-
27
Nhm cht Phn ng M t Dng tnh
mi cho n kh, ri
thm 1ml anhydrid
acetic, lc k. Thm 1ml
acid sulfuric c bng
cch nh theo thnh ng
nghing 45o.
mu t hng n
xanh l
Cht bo Vt trn giy lc
Dch chit, lc vi ether
du ha, nh 1 git ether
du ha ln giy lc, h
nng cho bay ht dung
mi.
Vt m trn giy
lc
2.3.2. Xy dng quy trnh phn tch nh tnh bng phng php sc k lp
mng
Cc mu l c tch chit phn on nh trnh by trong Hnh 1.3. Cc
phn on thu c s c phn tch nh tnh bng phng php sc k lp
mng. Trong , mt s yu t c nh hng n qu trnh phn tch, bao gm:
phng php chit xut v h dung mi pha ng, khong khai trin v th
tch
phun. Do vy, cn tin hnh nghin cu s nh hng ca cc yu t n kt qu
phn tch. Cc th nghim sng lc sau c tin hnh trn bt l Gymnema
latifolium Wall. ex Wight thu c ti Thi Nguyn.
2.3.2.1. Sng lc phng php chit xut:
Tin hnh chit 10g bt dc liu xay th CX1, CX2, CX3 bng 300ml
methanol tuyt i bng 3 phng php: Ngm lnh, Ngm kit, Hi lu thu
c
cc dch chit tng ng: NL, NK, HL. Mt lng nh dch chit thu c theo
3
phng php c dng chm sc k vi:
Bn mng trng sn Silica Gel GF254 (Merck) hot ha 1100C trong
1h.
H dung mi khai trin: chloroform : methanol : acid formic
(10:1,1:0,2).
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28
Hin mu UV 365nm hoc nhng thuc th hin mu dung dch acid
sulfuric 10%, h nng n khi hin mu.
Phn cn li em c thnh cn v cn bng cn k thut.
2.3.2.2.Sng lc h dung mi pha ng trong phn tch TLC
Tin hnh chit tch phn on dch chit Gymnema latifolium Wallich
ex
Wight (mu DM) theo quy trnh trnh by trong Hnh 1.3 vi phng php
chit
xut l phng php hi lu. nh tnh cc phn on dch chit bng cc phn
ng
thng quy trong Bng 2.2.
Bng 2.2 xc nh nhm cht chnh s tp trung phn tch. Tin hnh sng
lc cc h dung mi pha ng thng dng trong phn tch cc nhm cht v
chn ra h dung mi cho kt qu tch vt sc k tt nht.
Sc k lp mng (TLC): c thc hin trn bn mng trng sn TLC Silica
Gel 60 F254 (Merck); pht hin vt UV 366nm hoc dng thuc th l dung
dch
H2SO4 10%, sy 90oC n khi hin mu.
Da vo kt qu nh tnh v sng lc thu c, xut quy trnh phn tch
nh tnh cc phn on dch chit cc mu thu hi v m ha trong Bng 2.1.
2.3.3.Tin hnh phn tch nh tnh cc phn on dch chit mt s loi
trong
chi Gymnema R.Br. theo quy trnh xut
Tin hnh phn tch nh tnh cc mu theo quy trnh xut.
Xc nh cc vch c trng chi v vch c trng loi:
- Vch c trng chi l vch sc k xut hin tt c cc mu nghin cu
thuc chi Gymnema R.Br. khi tin hnh phn tch vi cng mt h dung
mi
pha ng v quan st, hin mu vi cng mt phng php.
- Vch c trng loi l vch sc k xut hin tt c cc mu nghien cu
thuc cng mt loi nhng khng c loi khc trong cng chi khi tin
hnh
phn tch vi cng mt h dung mi pha ng v quan st, hin mu vi cng
mt phng php.
c im v thnh phn ha hc ca cc mu c thng k qua kt qu sc
k thu c. S c mt ca cc vch trn sc k c m ha nh bin,
-
29
trong 1 l c vch v 0 l khng c vch. H s ng dng Sij ca cc mu
c tnh theo cng thc ca Nei & Li [24]:
Trong :
Sij : H s ng dng
Nij : S vch chung ca hai loi i v j
Ni : S vch ca loi i
Nj : S vch ca loi j
Trn c s bng h s ng dng vch trn sc k ca 12 mu nghin
cu, thit lp cy phn loi s dng php phn tch chm theo phng php
khong
cch kin kt trung bnh UPGMA (Unweighted Pair-Group Method
with
Arithmetical Averages), s dng phn mm NT-SYS PC 2.02h
(Numerical
Taxonomy System Applied Biostatistics, New York).
-
30
CHNG 3. THC NGHIM, KT QU V BN LUN
3.1. NH TNH CC NHM CHT CHNH
Kt qu nh tnh thc hin theo cc phn ng thng quy c trnh by
trong Bng 3.1.
Bng 3.1. Kt qu cc phn ng nh tnh thnh phn ha hc mt s loi
trong chi Gymnema R.Br
Nhm cht Phn ng GL1 GS2 GI1 GY1 GS1
Glycosid
tim
Phn ng Raymond
Marthoud
- - - - -
nh tnh ng 2-desoxy - - - - -
Saponin Phn ng Salkowski + + + + +
Phn ng to bt +++
Ct bt 2 ng cao bng nhau
Phn ng Rosenthaler +++ +++ +++ +++ +++
Flavonoid Phn ng Cyanidin +++ +++ +++ +++ +++
nh tnh anthocyanosid - - - - -
nh tnh proanthocyanidin - - - - -
Alkaloid Thuc th Dragendorff - - - - -
Thuc th Mayer - - - - -
Thuc th Wagner - - - - -
Anthranoid Phn ng Borntraeger - - - - -
Coumarin Phn ng m ng vng lacton + + + + +
Phn ng Diazo + + + + +
Tanin Phn ng vi FeCl3 + + + + +
Phn ng vi ch acetat ++ ++ ++ ++ ++
Phn ng vi dung dch gelatin - - - - -
Tinh du Cn c mi thm - - - - -
ng kh Phn ng vi thuc th Fehling ++ ++ ++ ++ ++
-
31
Acid hu c Phn ng vi Na2CO3 - ++ - - +
Acid amin Phn ng vi ninhydrin 3% ++ ++ ++ + -
Sterol Phn ng Libermann ++ ++ ++ ++ +
Cht bo Vt trn giy lc + + + + +
Ghi ch: (-): Phn ng m tnh (+): Phn ng dng tnh
(++): Phn ng r (+++): Phn ng rt r
Kt lun:
L ca c 4 loi: Gymnema inodorum (Lour.) Decne, Gymnema
latifolium
Wall. ex Wight, Gymnema yunnanense Tsiang.
L Gymnema sylvestre (Retz) R.Br. ex Schult. v acid amin.
L Gymnema sp. c cha saponin, flavonoid, coumarin, ng kh,
sterol,
cht bo, acid hu c.
3.2. QUY TRNH NH TNH BNG PHNG PHP SC K LP
MNG
3.2.1. Sng lc phng php chit xut
Khi lng cn thu c t cc phng php chit xut c th hin trong
Bng 3.2.
Bng 3.2.Kt qu khi lng cn thu c khi chit xut bng cc phng
php khc nhau
Phng php M (g) M0 (g) M (g)
Ngm lnh 188,45 187,93 0,52
Ngm kit 187,36 187,76 0,60
Hi lu 188,46 187,84 0,62
Ch thch: M: khi lng cc cha cn
Mo: khi lng cc khng cha cn
M: khi lng cn ton phn
Nh vy, phng php hi lu cho khi lng cn ln nht l 0,62g v
phng php ngm lnh cho khi lng cn thp nht l 0,52g.
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32
S dng dch chit theo 3 phng php tin hnh khai trin sc k lp
mng vi h sung mi (S.2), hin mu di UV 366nm thu. Kt qu thu c
cho
thy: khng c s khc bit nhiu trong sc k gia cc dch chit c chit
bng 3 phng php khc nhau (Hnh 1.3).
`
NL NK HL
Hnh 3.1. Sc k dch chit NL, NK, HL vi h dung mi (S.2), hin mu
di UV 365nm
Nh vy, mu c chit bng cc phng php khc nhau cho ra sc k
khng khc bit, vic tc ng nhit khng nh hng n sc k .
Kt lun: S dng phng php chit xut hi lu trong quy trnh phn tch
nh tnh.
3.2.2. nh tnh cc phn on dch chit
Cc phn on dch chit n-hexan, ethylacetat, n-butanol v nc ca
mu
Gymnema latifolium Wall. ex Wight c nh tnh bng cc phn ng ha
hc
thng quy ca saponin v flavonoid.
Kt qu: Saponin v flavonoid tp trung ch yu trong phn on
ethylacetat
v phn on n-butanol. Trong phn on n-hexan khng c saponin v
flavonoid.
Trong phn on nc, c saponin nhng t v khng c flavonoid (Bng
3.3).
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33
Bng 3.3. Tm tt kt qu nh tnh saponin v flavonoid trong cc phn
on dch chit
Nhm cht Phn ng
Phn on
n-hexan Ethylacetat n-butanol Nc
Saponin Phn ng Salkowski - +++ ++ +
Phn ng to bt - +++ + +
Phn ng Rosenthaler - +++ ++ +
Flavonoid Phn ng Cyanidin - ++ + -
nh tnh
anthocyanosid
- - - -
nh tnh
proanthocyanidin
- - - -
Ghi ch: (-): Phn ng m tnh (+): Phn ng dng tnh
(++): Phn ng r (+++): Phn ng rt r
Kt lun: Tp trung phn tch hai nhm cht chnh l saponin v
flavonoid
trong dch chit ton phn, phn on ethylacetat v phn on
n-butanol.
3.2.3. Sng lc h dung mi pha ng
3.2.3.1. Sapnonin
Tin hnh sc k lp mng dch chit ton phn ca cy Gymnema
latifolium
Wall. ex Wight vi cc h dung mi khai trin sau:
(S.1) Chloroform: methanol: nc (15:5:1)
(S.2) Chloroform:methanol:acid formic (10:1,1:0,2)
(S.3) Ethylacetat : methanol: nc (100:17:13)
(S.4) Chloroform: aceton: acid formic (3,3:1:0,02)
(S.5) Chloroform:aceton:acid formic (1:3,5:0,02)
(S.6) Chloroform:methanol:acid formic (4:1:1)
(S.7) Toluen:chloroform:methanol (2:7:1)
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34
Bn sc k khai trin c hin mu UV 365nm v nhng vo thuc th l
dung dch H2SO4 10%, sy 900C trong 15 pht.
Kt qu: H dung mi (S.2) cho kt qu sc k tch tt nht. (Hnh 3.2)
Kt lun: Dng h dung mi (S.2) trong quy trnh phn tch nh tnh
bng
HPTLC.
3.2.3.2. Flavonoid
Tin hnh sc k lp mng dch chit ton phn ca cy Gymnema
latifolium
Wall. ex Wight vi cc h dung mi khai trin sau:
(F.1) Benzen:ethylacetat (3:1)
(F.2) Toluen:chloroform:aceton (6:10:4)
(F.3) Chloroform: ethylacetat:acid formic (5:4:1)
(F.4) Ethylacetat: toluen: methanol (8:6:1)
(F.5) Ethylacetat: n-buthanol: acid acetic :nc (5:3:1:1)
(F.6) Ethylacetat: methanol (4:1)
(F.8) Chloroform:methanol:acid acetic (7:1:1)
(F.9) Benzen : aceton (9:1)
(F.10) Benzen:aceton (7:1)
(F.11) Benzen: aceton (17:3)
Bn sc k khai trin c hin mu UV 365nm.
Kt qu: H dung mi F11 cho kt qu sc k tch tt nht (Hnh 3.2).
Kt lun: Dng h dung mi F.11 trong quy trnh phn tch nh tnh bng
TLC.
T cc kt qu sng lc nh trn, quy trnh nh tnh thnh phn ha hc mt
s loi trong chi Gymnema R.Br. c xut nh sau:
Chun b mu phn tch: i vi mi mu nghin cu, b phn s dng l
l, c sy kh 50-60oC, xay th. 50g dc liu c chit vi 500ml
methanol
tuyt i bng phng php hi lu trong 3 gi v lc qua giy lc thu c c
dch chit methanol. Dch chit ny c c thu hi dung mi bng my ct
quay
Buchi v th tch 100ml, thu c dch chit ton phn v lu 2ml dch chit c
c
-
35
s dng trong phn tch HPTLC. Lng dch chit c c cn li lc ln lt
vi
n-hexan (250ml). Lp methanol nc thu c c n cn ri hoa tan li
trong
100ml nc ct 1 ln thu c dch chit nc. Ln lt lc dch chit nc ny
vi
ethylacetat (250ml) v n-butanol (250ml) bo ha nc thu c cc phn
on
dch chit tng ng l: phn on n-hexan, phn on ethylacetat, phn on
n-
butanol v phn on nc. Cc phn on ethylacetat v phn on n-butanol
c
bc hi dung mi n cn, ri ha tan li trong methanol tuyt i thu c
dch
methanol ca cc phn on tng ng v c s dng trong phn tch HPTLC
(Hnh 1.3).
Sc k trong h F.11 Sc k trong h S.2
Hnh 3.2. Sc k mu DM vi h dung mi F.11 v S.2 hin mu di n
UV 365nm
Tin hnh phn tch HPTLC: Quy trnh phn tch nh tnh bng HPTLC
c tin hnh trn h thng HPTLC ca CAMAG, c m t nh trong mc
2.1.2. Cc mu phn tch c khai trin sc k vi hai h dung mi l (S.2)
v
(F.11), trn bn mng Silica Gel GF254 (Merck), khong khai trin
80mm. Bn
-
36
mng sau khi khai trin c chp nh cc bc sng UV 254nm, UV 366nm
v
hin mu bng cch nhng vo dung dch acid sulfuric 10%, sy 90oC trong
15
pht.
Hnh 3.3. Quy trnh phn tch nh tnh cc phn on dch chit bng
HPTLC
Dc liu
Dch chit
Dch chit c c
Phn on n-hexan Lp nc
Phn on
ethyl acetat Lp nc
Phn on n-butanol Phn on nc
Methanol tuyt i
C cch thy
N-hexan
Ethyl acetat
N-butanol
bo ha
nc
Cn E
Cn B
HPTLC
Chit hi lu
-
37
3.3. Phn tch nh tnh cc dch chit ton phn v phn on dch chit
theo
quy trnh xut
Cc mu c m ha trong Bng 2.1 c tin hnh x l nh trong quy trnh m
t mc 2.3.2 (
Hnh 3.3). Kt qu thu c nh sau:
3.3.1.1. Sc k
Dch chit ton phn, phn on ethylacetat, phn on n-butanol c
khai
trin sc k vi hai h dung mi l S.2 v F.11 thu c 6 bn sc k . Kt
qu
cho thy cc phn on dch chit tch tt vi h dung mi S.2 v tch km hn
vi
h dung mi F.11. Dch chit ton phn v phn on ethylacetat cho nhiu
vch
sc k i vi c hai h dung mi, trong c nhiu vch xut hin tt c cc
mu
(vch c trng chi) v mt vi vch ch xut hin nhng mu cng loi (vch
c
trng loi). Phn on n-butanol c nhiu s khc bit nht gia cc mu,
trong ,
mu GL3, GS2 v GY1 khng xut hin vch no di UV 366nm v khi hin
mu
bng dung dch acid sulfuric 10%. Do vy, vic xc nh cc vt c trng da
trn
sc k ca dch chit ton phn v phn on ethylacetat.
Vch c trng chi: Da trn sc k Hnh 3.4 v Hnh 3.5, quan st c
cc vch sc k xut hin tt c cc mu, l cc vch c trng chi. Gi tr
Rf
ca cc vch ny c th hin trong Bng 3.4.
Bng 3.4. Gi tr Rf ca cc vch c trng chi Gymnema R. Br.
H dung mi
pha ng
Quan st Phn on dch chit khai trin
Dch chit ton phn Phn on ethylacetat
H S.2 UV 254nm 0,89; 0,84 0,64
UV 366nm 0,47 0,88; 0,66; 0,47
VIS H2SO4 0,46; 0,25 0,20; 0,66
H F.11 UV 254nm Khng c Khng c
UV 366nm 0,59; 0,49 0,59; 0,49
VIS H2SO4 0.55 Khng c
-
38
Hnh 3.4. Sc k khai trin dch chit ton phn v phn on
ethylacetat
vi h dung mi S.2
-
39
Hnh 3.5. Sc k khai trin dch chit ton phn v phn on
ethylacetat
vi h dung mi F.11
-
40
Vch c trng loi: T cc sc k c th xc nh c mt s vch sc
k c trng cho cc loi nghin cu.
1. Gymnema sylvestre (Retz.) R.Br. ex Schult.:
Vch c trng loi: Trn sc k khai trin dch chit ton phn vi h
dung mi S.2 v quan st di UV 254nm, cc vch sc k ti Rf 0,31 v 0,06
ch
xut hin 4 mu thuc loi Gymnema sylvestre (Retz.) R.Br. ex
Schult.. Khi hin
mu bng dung dch acid sulfuric 10% th xut hin vch sc k c trng cho
loi
Gymnema sylvestre (Retz.) R.Br. ex Schult. Rf 0,14. Trn sc k
khai trin
phn on ethylacetat vi h dung mi S.2 v hin mu bng dung dch
acid
sulfuric 10% th quan st c mt nhm vch c trng ti Rf 0,66; 0,57;
0,51;
0,47; 0,42 v 0,09 (Hnh 3.4).
S khc bit gia cc mu trong cng mt loi: Cc mu ca loi Gymnema
sylvestre (Retz.) R.Br. ex Schult. c s tng ng ln. Tuy nhin, trn
bn sc k
khai trin dch chit ton phn vi h dung mi S.2, hin mu bng dung
dch
acid sulfuric 10%, vch sc k ti Rf 0.63 xut hin c 3 mu GS3, GS4,
GS5
nhng khng c mu GS2 (Hnh 3.6). Nh vy, tn ti s khc bit gia mu
GS2
vi 3 mu GS3, GS4, GS5.
2. Gymnema inodorum (Lour.) Decnc:
Vch c trng loi: Trn sc k khai trin dch chit ton phn vi h
dung mi S.2 v quan st UV 254nm v UV 366nm, quan st c vch sc k
c
hunh quang mu xanh Rf 0,07 rt c trng ca 2 mu loi Gymnema
inodorum
(Lour.) Decnc. Vch sc k ny cng xut hin sc k phn on
ethylacetat
vi h dung mi S.2 ca 2 mu loi Gymnema inodorum (Lour.) Decnc.
(Hnh 3.4).
Trn sc k khai trin phn on ethylacetat vi h dung S.2, di UV
366nm
cn xut hin vch c hunh quang xanh ti Rf 0,55 ch c 2 mu ca loi
Gymnema inodorum (Lour.) Decnc. (Hnh 3.4). Vi h dung mi F.11,
trn bn sc
k hin mu bng dung dch acid sulfuric 10%, dch chit ton phn ca 2
mu GI1
v GI2 cho 2 vch sc k ti Rf 0,61 v 0,58 thay v 1 vch sc k ti Rf
0,60 nh
cc mu cn li (Hnh 3.5).
-
41
S khc bit gia cc mu trong loi: Trn sc k khai trin dch chit
ton phn vi h dung mi S.2, quan st UV 254nm v hin mu bng dung
dch
acid sulfuric 10%, vch sc k Rf 0,75 xut hin mu GI1 nhng khng
c
mu GI2 (Hnh 3.7 a, b). Trn sc k khai trin phn on ethylacetat vi
h
dung mi S.2, quan st UV 254nm v hin mu bng dung dch acid
sulfuric 10%,
2 vch sc k Rf 0,69 v 0,50 cng c mu GI1 nhng khng c mu GI2
(Hnh 3.7 c,d). Vi h dung mi F.11, hin mu bng dung dch acid
sulfuric
10%, vch sc k ti Rf 0,26 v 0,22 xut hin dch chit ton phn GI1
nhng
khng c mu GI2. Ngc li, dch chit ton phn GI2 cho vch mu xanh ti
Rf
0,47 m mu GI1 khng c (Hnh 3.7 e).
3. Gymnema yunnanense Tsiang:
T cc bn sc k , quan st thy khng c vch sc k no ch xut hin
mu Gymnema yunnanense Tsiang, nhng sc k ca mu GY1 cng khng
ging hon ton vi cc mu cn li. Do , da vo sc k vn c kh nng
phn bit mu GY1 vi cc mu cn li.
4. Gymnema latifolium Wallich ex Wight.:
Ging nh loi Gymnema yunnanense Tsiang, da vo sc k , khng
nhn thy c vch sc k c trng cho loi. T sc k , 4 mu Gymnema
latifolium Wallich ex Wight. c s khc bit nht nh mt s vch sc k.
Vi h
dung mi S.2, quan st UV 366nm, dch chit ton phn ca 3 mu GL1,
GL3,
GL4 cho vch sc k m ti Rf 0,58 nhng vch ny khng xut hin mu
GL2.
Trong khi , ti v tr Rf 0,35, vch mu xanh xut hin mu GL2 nhng
khng
c cc mu cn li (Hnh 3.8 a). Khi hin mu bn ny bng dung dch
acid
sulfuric 10%, GL4 cho vch sc k mu xanh tm ti Rf 0,61 trong khi 3
mu cn
li khng c (Hnh 3.8 b). Cng vi h dung mi ny, phn on ethylacetat
ca
c 4 mu u cho vch sc k ti Rf 0,35 v 0,27 nhng mu sc cc vch li
khc
nhau. 4 vch ny ca mu GL3 v GL4 u c mu , trong khi , vch sc k
ti
Rf 0,27 ca mu GL1 v GL2 c mu xanh v vch sc k ti Rf 0,35 c mu
xanh
dng (Hnh 3.8 c).
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42
5. Gymnema sp.:
Vi h dung mi S.2, quan st UV 366nm, c dch chit ton phn v phn
on ethylacetat u cho vch sc k hunh quang xanh da tri ti Rf 0,37
(Hnh
3.4). Vi h dung mi F.11, quan st UV 366nm, c dch chit ton phn v
phn
on ethylacetat u cho vch sc k hunh quang xanh Rf 0,25 (Hnh
3.5).
Hnh 3.6. S khc bit gia cc mu ca loi Gymnema sylvestre (Retz.)
R.Br. ex
Schult
-
43
Hnh 3.7. S khc nhau gia cc mu ca loi Gymnema inodorum
(Lour.)
Decnc.
Hnh 3.8.S khc nhau gia cc mu ca loi Gymnema latifolium
Wallich
ex Wight.
3.3.1.2. H s tng ng gia cc mu nghin cu
Kt qu khai trin sc k t 3 phn on dch chit ca 12 mu vi hai h
dung mi S.2 v F.11 c 252 vch sc k trong vi m nht khc nhau.
Cc
-
44
vch sc k c m ha nh bin lp thnh bng (Ph lc). Mi tng quan v
thnh phn ha hc c th hin qua h s tng ng cp i gia cc mu
nghin cu, s dng ch s Nei&Li (Bng 3.5). H s tng ng c gi tr t
0,51
0,94. Trong , gi tr tng ng cao nht 0,94 c 2 mu GS4 v GS5 c
trng cng mt a im l Thi Nguyn v thu hi cng mt thi im.
Bng 3.5. Bng h s tng ng cp i gia cc mu nghin cu, s dng
ch s Nei&Li
GL1 GL2 GL3 GL4 GS1 GS2 GS3 GS4 GS5 GI1 GI2 GY1
GL1 1,00
GL2 0,80 1,00
GL3 0,89 0,79 1,00
GL4 0,76 0,78 0,79 1,00
GS1 0,72 0,68 0,71 0,62 1,00
GS2 0,66 0,60 0,66 0,62 0,62 1,00
GS3 0,66 0,64 0,66 0,70 0,62 0,86 1,00
GS4 0,61 0,60 0,64 0,68 0,62 0,82 0,89 1,00
GS5 0,62 0,60 0,65 0,66 0,59 0,81 0,86 0,94 1,00
GI1 0,61 0,57 0,62 0,65 0,51 0,69 0,64 0,60 0,60 1,00
GI2 0,58 0,59 0,60 0,69 0,60 0,60 0,64 0,65 0,62 0,72 1,00
GY1 0,71 0,65 0,71 0,65 0,70 0,70 0,62 0,61 0,60 0,67 0,65
1,00
3.3.1.3. Cy phn loi
Cy phn loi cc mu c trnh by trong Hnh 3.9. mc tng ng
cao hn 70%, cc mu thuc loi Gymnema R. Br. trong nghin cu c
chia
thnh cc nhm:
Nhm (I) tng ng v mt hnh thi l cc mu thuc loi Gymnema
latifolium Wall. Ex Wight.. Trong , mu GL1 (thu hi Thi Nguyn)
v
GL3 (thu hi Gia Lai) c h s tng ng cao hn 80%, sau l GL2 (thu
-
45
hi Ty Ninh) vi h s tng ng gn 80% v GL4 c nhiu khc bit nht
vi cc mu khc trong loi vi h s tng ng khong 78%.
Nhm (II) l mu GS1, mu Gymnema sp. thu hi CHDCND Lo.
Nhm (III) l mu GY1, mu Gymnema yunnanense Tsiang.
Nhm (IV) bao gm cc mu thuc loi Gymnema sylvestre (Retz.) R. Br.
ex
Trong nhm ny, mu GS4 (mu hoa vng) v GS5 (mu hoa ), u c
thu hi Thi Nguyn, t h s tng ng cao nht 94%, tc l hu nh khng
c s khc bit v v tr cc vch sc k. Mu GS3 c nhiu im tng ng
ha hc vi GS4 v GS5 hn so vi mu GS2 mc d GS3 v GS2 u l mu
hoa trng nhng c trng ti hai a im khc nhau l Thi Nguyn v
Thanh Ha.
Nhm (V) bao gm 2 mu GI1 v GI2 thuc loi Gymnema inodorum
(Lour.)
Decne. H s tng ng ca hai mu thp hn so vi cc nhm khc (khong
70%).
Hnh 3.9. Cy phn loi da trn phn tch UPGMA h s tng ng gia
sc k ca cc mu
-
46
3.4. BN LUN
3.4.1. V phng php nghin cu
Chemotaxonomy l phn loi thc vt da trn phn tch thnh phn ha hc
ca cc dc liu. Trong vi thp nin tr li y, phn tch thnh phn cc
sn
phn c ngun gc thin nhin c n gin ha bng vic khai trin sc k v
in di. C s ca phng php l s dng phng php sc k lp mng hoc sc
k lp mng hiu nng cao vi cc iu kin ti u phn tch thnh phn ha
hc
ca cc loi thc vt, t xc nh cc vch sc k c trng cho tng chi v
tng
loi v xy dng nn cy phn loi thc vt da trn thnh phn ha hc. Cho
n
nay, nhiu nghin cu c thc hin trn mt s chi thc vt nh Hypericum
L.
[19], Papaver L. [40], v.v
Ngy nay, HPTLC c s dng trong nghin cu phn loi cc loi v di
loi (chemotype) bi s chun ha tt c cc bc. Cc thng s k thut nh
hng ln n qu trnh khai trin TLC nh khong khai trin, th tch phun
mu,
thi gian sy, thi gian bo ha dung mi, v.v u c chun ha thng
qua
phn mm my tnh ng thi cc thng s iu kin tin hnh nh nhit , m,
v.v cng c ghi li, m bo chnh xc v lp li cao nht. Bn cnh vic
ng dng trong phn loi thc vt da trn thnh phn ha hc, HPTLC cn
c
s dng chun ha quy trnh chit xut cng nh kim nh cc thuc c ngun
gc t thin nhin. Dc in Trung Quc ng dng phng php ny xy
dng nn c s d liu kim nh thuc c ngun gc t thin nhin [18]. Vi
cc
u im trn, nghin cu ny s dng phng php HPTLC nhm bc u xc
nh c Rf ca cc marker ha hc c trng ca cc loi thuc chi Gymnema
R.Br. cng nh l s khc nhau gia cc mu thuc cng mt loi nhng khc
nhau
v a l.
3.4.2.V nh tnh cc nhm cht chnh
Trong phn tch nh tnh thnh phn ha hc trong cc mu dc liu, vic
nh tnh cc nhm cht chnh bng cc phn ng ha hc c trng l cn thit,
gp phn hiu bit s b v thnh phn ha hc chnh v nh hng nghin cu.
-
47
Kt qu nghin cu cho thy l ca 5 loi Gymnema inodorum (Lour.)
Decne, Gymnema latifolium Wall. Ex Wight, Gymnema yunnanense
Tsiang,
sylvestre (Retz) R. Br. ex Schult., Gymnema sp. c cc nhm cht
ging nhau.
l saponin, flavonoid, coumarin, ng kh, acid amin, sterol v cht
bo. Ngoi
cc nhm cht trn, l Gymnema sylvestre (Retz) R. Br. ex Schult. cn
c acid hu
c. Trong khi , l Gymnema sp. thu hi ti CHDCND Lo li m tnh vi
phi
ng ca acid hu c. V c bn, kt qu ny ph hp vi kt qu nghin cu
c cng b trc y [12], [7].
Bn cnh , phn ng ca saponin v flavonoid cho kt qu dng tnh thy
r nht. ng thi, hai nhm cht ny ng vai tr quan trng trong tc dng
sinh
hc ca Gymnema sylvestre (Retz) R. Br. ex Schult. [36], [41],
[27], [28] .Do ,
quy trnh nh tnh TLC xy dng cc bc sau s tp trung ch yu vo hai
nhm cht ny.
3.4.3. V xy dng quy trnh nh tnh bng HPTLC
m bo chnh xc v lp li th vic sng lc cc yu t nh hng
chnh xy dng quy trnh nh tnh l cn thit c c kt qu ti u.
V phng php chit xut: Phng php hi lu cho khi lng cn ln
nht trong 3 phng php chit xut th nghim. Kt qu kho st thc nghim
cho
thy vic tc ng nhit (50-60oC) khng nh hng n s n nh cc thnh
phn
ha hc trong dch chit. Ngoi ra, xt v hiu qu thi gian, vic tc ng
nhit y
nhanh qu trnh chit xut v gip tit kim thi gian hn phng php ngm
lnh
v ngm kit. V vy, vic la chn phng php hi lu l hp l, tuy nhin
cn
lu kim sot nhit , duy tr trong khong 50-60oC trong qu trnh gia
nhit.
V nh tnh cc nhm cht chnh c trong cc phn on dch chit: Saponin
v flavonoid ch yu tp trung trong phn on ethylacetat v n-butanol.
Trong
phn on n-hexan khng c saponin v flavonoid; phn on nc ch cn
saponin
vi lng nh (phn ng dng tnh khng r). iu ny gip nghin cu tp
trung
vo hai phn on ethylacetat v n-butanol cng vi dch chit ton phn, v
bc
u khu tr cc h dung mi c th s dng phn tch TLC.
-
48
V h dung mi pha ng: H dung mi pha ng l yu t quan trng quyt
nh phn gii ca kt qu sc k . Da vo kt qu nh tnh, vic sng lc h
dung mi pha ng c tp trung ch yu vo h ca hai nhm cht chnh cn
phn tch l saponin v flavonoid. H dung mi pha ng l mt trong nhng
yu t
quan trng nht tc ng n kt qu sc k . Do , vic la chn c h dung
mi cho phn gii cao l iu cn thit. Kt qu cho thy, h dung mi
S.2
chloroform:methanol:acid formic (10:1,1:0,2) v h dung mi F.11
benzen:aceton
(34:6) cho kt qu tch tt nht.
Nh vy, nghin cu bc u sng lc c mt s yu t nh hng
chnh n phn tch nh tnh TLC, tuy nhin cn tn ti nhiu yu t nh
hng
khc nh khong khai trin, th tch mu chm... cn c sng lc c c quy
trnh ti u nht.
3.4.4. V phn tch nh tnh cc mu theo quy trnh xut
a. Cc phn on ha hc nghin cu:
Qu trnh phn tch cho kt qu l 6 sc k . Sc k ca dch chit ton
phn v phn on ethylaceatat cho nhiu vch sc k. Trn sc k phn on
ethylacetat, cc vch sc k xut hin r rng v gn hn, t vch sc k hn so
vi
dch chit ton phn. iu ny cho thy vic lc dch chit ton phn vi
n-hexan
gip loi b c nhiu tp cht, lm cho sc k phn on ethylacetat sch
s
v r rng hn. Nh vy, vic x l mu trc khi phn tch HPTLC l cn
thit
c c kt qu tt nht. i vi phn on n-butanol, cc vch sc k xut hin
t
hn nhiu, v ti cc v tr khc nhau, khng c quy lut. c bit, mu GL3,
GS2
v GI1 hu nh khng cn vt sc k no. Gi thuyt a ra l gii iu ny l
cc hp cht c trong dch chit, sau khi lc vi ethylacetat, tan ht vo
phn
on ny, do phn on b-butanol hu nh khng cho vch sc k no; cc mu
khc vn cho vch sc k phn on n-butanol l do th tch ethylacetat
cha
ha tan ht cc hp cht c trong dch chit. y cng l mt c im khc
bit
phn bit cc mu, song, do trong nghin cu, vic kim sot th tch v
nng
-
49
cha cht ch, nn khng a ra kt lun vi phn on n-butanol. Cn tin
hnh
mt nghin cu khc c th a ra kt lun chnh xc.
b. Xc nh cc vch c trng chi v vch c trng loi:
T kt qu sc k xc nh c cc vch c trng chi v vch c trng
loi c lit k trong mc 3.3.1.1. Vic tin hnh phn tch mt mu bt dc
liu
cha bit tn theo quy trnh xut trn v i chiu vi cc vch c trng chi
v
vch c trng loi c th gip s b xc nh bt dc liu c thuc chi
Gymnema R. Br. hay khng. Cng vi cc bng chng khc v di truyn v
tc
dng sinh hc, c th khng nh ngun gc bt dc liu. Bn cnh , vic
phn
lp v xc nh cu trc ca cc vch c trng c th gip pht trin cht
chun,
hay cn gi l ch th ha hc ca chi Gymnema R. Br. v cc loi.
Bn cnh cc vch c trng cho chi v cho loi, cc loi trong cng mt
chi
cng c nhng c im sc k khc nhau. S khc bit c th c m t trong
mc 3.3.1.1. Nhng s khc bit ny cho php phn bit cc loi trong cng
mt chi
bng phng php TLC. Bn thn trong cng mt loi, cc mu c thu hi
nhng a im khc nhau cng nh thi im thu hi khc nhau cng cho c
im
sc k khc nhau. Thc t, yu t kh hu a cht c nh hng ln ti thnh
phn ha hc trong cy c. iu ny c chng minh s khc bit gia 4 mu
Gymnema sylvestre (Retz) R. Br. ex Schult. nghin cu. Mu GS2 v
GS3 ging
nhau hon ton v mt hnh thi thc vt (u l Gymnema sylvestre (Retz)
R. Br.
ex Schult. hoa trng) song vn tn ti nhng s khc bit trong sc k gia
hai
mu ny. Trong khi , GS3, GS4, GS5 cng thuc loi Gymnema sylvestre
(Retz.)
R.Br. ex Schult. nhng c s khc bit v hnh thi l mu hoa nhng GS3 li
c
nhiu im tng ng vi GS4 hn so vi GS2. C th, h s tng ng gia
GS3 vi GS4 l 0,89 trong khi h s tng ng gia GS3 vi GS2 l 0,86.
iu
ny c th c l gii do iu kin nui trng v thu hi. Ba mu GS3, GS4,
GS5
c thu hi cng mt a im (Thi Nguyn) vo cng mt thi im (thng
5/2013), trong khi mu GS2 c thu hi Thanh Ha v vo thng
4/2013.
-
50
Tng t vi cc mu thuc loi Gymnema latifolium Wallich ex
Wight..
Bn mu c thu hi ti 4 a im khc nhau v vo thi im khc nhau (Bng
2.1) do tn ti nhiu s khc bit gia sc k ca 4 mu trong cng iu
kin
phn tch. iu ng ch l mu GL1 c thu hi ti Thi Nguyn v mu GL3
c thu hi ti Ha Bnh. Trn thc t, ging cy Gymnema latifolium
Wallich ex
Wight. Ha Bnh c ngun gc Thi Nguyn nhng sc k cho thy tn ti
nhiu khc bit gia hai mu ny (mc 3.3.1.1) vi h s tng ng l 0,76,
thp
hn so vi h s tng ng ca GL1 vi GL2 (0,80) v GL2 vi GL3
(0,89).
Nhng kt qu trn cho thy nu c nh iu kin nui trng v thi gian
thu hi, th cc mu thuc cng mt loi s c thnh phn ha hc n nh v t
hiu qu cao trong vic xc nh chnh xc cc chemotype thc vt. iu ny
cng
c ngha khi mun pht trin mt loi cy thuc trn mt a im mi, cn
phi
tin hnh cc nghin cu xc nh iu kin nui trng v thu hi ti u thu
c dc liu cht lng nht.
c. Xy dng cy phn loi:
Cy phn loi xy dng da trn h s tng ng gp phn khng nh
nguyn l l cc loi cng mt bc taxon thc vt thng c cc thnh phn ha
hc
tng t nhau do c xu hng c tc dng sinh hc ging nhau. Bn cnh ,
s
khc bit v h s tng ng gia cc nhm l kh ln (>30%) cho thy hon
ton
c th s dng ch th ha hc phn bit cc mu mc loi. Cc mu thuc
cng mt loi u nm trong cng mt nhm tng ng ha hc vi nhau. C th
l: Nhm I l cc mu thuc loi Gymnema latifolium Wall. Ex Wight, nhm
II l
mu cha xc nh tn khoa hc thu hi ti CHDCND Lo, nhm III l mu
thuc
Gymnema yunnanense Tsiang, nhm IV l cc mu thuc loi Gymnema
sylvestre
(Retz) R. Br. ex Schult. v nhm V l cc mu thuc loi Gymnema
inodorum
(Lour.) Decne. c bit, mu Gymnema sp. thu hi ti CHDCND Lo mc d
c
c im hnh thi v c im di truyn cng nhm vi G.sylvestre (Retz) R.
Br.
ex Schult. [13] nhng li c h s tng ng vi mu Gymnema
yunnanense
Tsiang cao hn. Kt qu ny b sung thm c s khng nh s khc bit mc
-
51
loi ca mu Gymnema sp.. Do vy, cn c thm nghin cu khc tin hnh
xc
nh tn khoa hc ca mu ny. Vi h s tng ng hn 70%, cc mu nghin
cu c thnh phn ha hc tng i ging nhau, v vy hon ton c kh nng
hot
cht c tc dng sinh hc loi ny cng c loi kia. iu ny hon ton ph
hp
vi cc nghin cu tin hnh trc , chng minh Gymnema latifolium Wall.
ex
Wight, Gymnema yunnanense Tsiang v Gymnema inodorum (Lour.)
Decne cng
c tc dng h ng huyt ging nh Gymnema sylvestre (Retz) R. Br. ex
Schult..
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52
KT LUN
1. V nh tnh cc nhm cht chnh trong mt s loi thuc chi Gymnema R.
Br.
- L Gymnema inodorum (Lour.) Decne, l Gymnema latifolium Wall.
ex
Wight, l Gymnema yunnanense Tsiang u c cha saponin,
flavonoid,
coumarin, ng kh, sterol, cht bo v acid amin.
- L Gymnema sylvestre (Retz) R. Br. ex Schult. c cha saponin,
flavonoid,
coumarin, ng kh, sterol, cht bo v acid hu c.
Trong , thnh phn saponin l saponin triterpenoid v thnh phn
flavonoid l cc
flavonoid nhm -pyron v -dihydropyron.
2. xy dng v m t c quy trnh phn tch nh tnh cc loi thuc chi
Gymnema R. Br. vi cc iu kin: Phng php chit xut hi lu, h dung
mi
pha ng.
3. nh tnh thnh phn ha hc mt s mu cy thuc chi Gymnema R. Br. thu
hi
Vit Nam v xc nh c h s ng dng ha hc ca mt s loi thc chi
c gi tr 0,51 0,94. h s ng dng ha hc 70%, cc mu nghin cu chia
thnh 5 nhm tng ng vi s phn chia loi hnh thi. Thnh phn ha hc
ca cc cht c s khc nhau gia 5 mu c trng c nh ti mt a im
thu hi vo cng mt thi gian do c th d on c s khc nhau v bn cht
di truyn ca cc mu cy da trn c im ha hc.
XUT
1. Tip tc nghin cu s a dng loi da trn dch chit thy phn.
2. Nghin cu xc nh cu trc ca cc vch c trng chi xc nh thnh phn
ha hc ch th c trng chung ca chi Gymnema R.Br.
3. Nghin cu xc nh cu trc ca cc vch c trng loi xc nh thnh
phn ha hc c trng phn bit gia cc loi khc nhau trong chi
Gymnema
R.Br.
-
53
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review of herbs and dietary supplements for glycemic control in
diabetes",
Diabetes care, 26, pp. 1277-1294.
44. Xie J. T., Wang A., Mehendale S., Wu J., Aung H. H., Dey L.,
et al. (2003),
"Anti-diabetic effects of Gymnema yunnanense extract",
Pharmacological
research : the official journal of the Italian Pharmacological
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47(323-329).
-
PH LC: Bng m ha nh bin cc vch sc k
I. Khai trin dch chit ton phn vi h dung mi S.2
Bng I.1. Quan st UV 254nm
Rf GL1 GL2 GL3 GL4 GS1 GS2 GS3 GS4 GS5 GI1 GI2 GY1
0.89 1 1 1 1 1 1 1 1 1 1 1 1
0.84 1 1 1 1 1 1 1 1 1 1 1 1
0.75 0 1 0 1 0 0 0 0 0 1 0 0
0.63 0 0 0 1 0 1 1 1 1 1 1 0
0.58 1 0 1 1 1 1 1 1 1 1 1 1
0.52 0 0 0 1 0 0 0 0 0 1 1 0
0.45 1 1 1 1 1 1 1 1 1 1 0 1
0.43 1 0 0 0 0 0 0 0 0 0 0 1
0.38 1 0 1 1 0 1 1 1 1 0 0 1
0.33 0 0 0 0 0 1 1 1 1 0 0 0
0.26 1 0 1 1 0 1 1 1 1 1 0 0
0.20 1 0 1 1 0 1 1 1 1 1 0 0
0.14 0 0 0 0 0 1 1 1 1 0 0 0
0.12 0 0 0 0 0 1 1 1 1 0 0 0
0.08 0 0 1 1 0 0 0 0 0 1 1 1
0.06 0 0 0 0 0 1 1 1 1 0 0 0
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Bng I.2. Quan st UV 366nm
Rf GL1 GL2 GL3 Gl4 GS1 GS2 GS3 GS4 GS5 GI1 GI2 GY1
0.89 1 1 1 1 1 1 1 1 1 1 1 0
0.86 0 0 0 0 0 0 0 0 0 0 0 1
0.84 1 1 1 1 1 1 1 1 1 1 1 0
0.76 1 1 1 1 1 1 1 1 1 1 1 1
0.72 0 1 0 1 1 1 1 1 1 1 1 1
0.63 0 0 0 0 0 1 0 0 0 1 0 0
0.59 1 0 1 1 1 1 1 1 1 1 1 1
0.56 0 0 0 0 0 0 0 0 0 1 1 0
0.48 0 0 0 0 0 0 0 0 0 1 1 0
0.47 1 1 1 1 1 1 1 1 1 1 1 1
0.40 1 0 1 1 1 1 1 1 1 1 1 1
0.38 1 0 1 1 1 1 1 1 1 1 1 1
0.36 0 0 0 0 0 1 1 1 1 1 1 1
0.32 0 0 0 0 1 1 1 1 1 1 1 1
0.21 0 0 0 0 0 1 0 1 0 0 0 0
0.18 0 0 0 0 0 1 1 1 1 1 1 0
0.13 0 0 0 0 0 0 0 0 0 1 1 0
0.11 1 1 1 1 1 0 0 0 0 0 0 1
0.08 0 0 0 0 0 0 0 0 0 0 0 0
0.04 1 1 1 1 0 0 0 0 0 1 1 1
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Bng I.3. Hin mu vi dung dch acid sulfuric 10% v quan st nh sng
trng
Rf GL1 GL2 GL3 GL4 GS1 GS2 GS3 GS4 GS5 GI1 GI2 GY1
0.89 0 0 0 0 0 1 1 1 1 1 1 1
0.86 1 1 1 1 1 0 0 0 0 0 0 1
0.84 0 0 0 1 0 1 1 1 1 1 1 0
0.80 1 1 1 1 1 1 1 1 1 0 0 1
0.75 0 1 0 1 0 1 1 1 1 1 0 0
0.72 1 1 1 1 1 1 1 1 1 1 0 1
0.63 0 0 0 1 0 0 1 1 1 1 1 0
0.59 1 0 1 1 1 1 1 1 1 1 1 1
0.52 0 0 0 0 0 1 1 0 0 1 0 1
0.46 1 1 1 1 1 1 1 1 1 1 1 1
0.42 1 1 1 1 0 1 1 1 1 1 1 1
0.38 0 0 0 1 0 1 0 0 0 1 1 1
0.25 1 1 1 1 1 1 1 1 1 1 1 1
0.14 0 0 0 0 0 1 1 1 1 0 0 1
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II. Khai trin phn on dch chit ethylacetat vi h dung mi S.2
Bng II.1. Quan st UV 254nm
Rf GL1 GL2 GL3 GL4 GS1 GS2 GS3 GS4 GS5 GI1 GI2 GY1
0.85 1 0 0 0 0 0 0 0 0 0 0 0
0.80 1 1 1 1 0 1 1 1 1 1 1 1
0.64 0 0 0 0 0 0 0 0 0 1 0 0
0.62 1 1 1 1 1 1 1 1 1 1 1 1
0.58 0 0 0 0 0 0 0 0 0 1 1 1
0.52 1 1 1 0 1 1 1 0 0 0 0 1
0.49 1 1 1 0 1 1 1 1 1 1 1 1
0.43 1 1 1 0 1 0 0 0 0 0 0 1
0.40 1 1 1 1 1 1 1 1 1 0 0 1
0.36 1 0 0 0 1 0 0 0 0 0 0 0
0.30 0 0 0 0 0 1 1 1 1 0 0 0
0.24 1 1 1 1 0 1 1 0 1 0 0 0
0.15 0 0 0 0 0 0 0 0 0 1 0 0
0.11 0 0 0 0 0 1 1 0 1 0 0 0
0.08 0 0 1 0 0 0 0 0 0 1 1 0
0.05 0 0 0 0 0 1 1 1 1 1 0 0
-
Bng II.2. Quan st UV 366nm
Rf GL1 GL2 GL3 GL4 GS1 GS2 GS3 GS4 GS5 GI1 GI2 GY1
0.86 1 1 1 0 0 1 0 0 1 1 0 0
0.80 1 1 1 1 1 1 1 1 1 1 1 1
0.74 1 1 1 1 1 1 1 0 0 1 0 1
0.70 0 0 0 0 0 0 0 0 0 1 1 0
0.62 1 1 1 1 1 1 1 1 1 1 1 1
0.58 1 1 1 0 1 1 1 1 0 1 1 0
0.52 0 0 0 0 0 0 0 0 0 1 1 0
0.49 1 1 1 1 1 1 1 1 1 1 1 1
0.41 1 1 1 1 1 1 1 1 1 1 0 0
0.33 1 1 0 0 1 1 1 0 0 1 1 1
0.23 1 1 1 1 1 1 1 1 1 1 1 1
0.16 0 0 0 0 1 1 1 0 0 1 1 1
0.13 1 1 1 1 1 1 0 0 0 1 1 0
0.12 1 1 1 1 0 0 0 0 0 1 1 0
0.07 1 1 1 1 0 0 0 0 0 1 1 0
-
Bng II.3. Hin mu bng thuc th acid sulfuric 10% v quan st nh sng
trng
Rf GL1 GL2 GL3 GL4 GS1 GS2 GS3 GS4 GS5 GI1 GI2 GY1
0.86 1 1 1 0 0 1 0 0 1 1 0 0
0.80 1 1 1 1 1 1 1 1 1 1 1 1
0.74 1 1 0 0 0 0 0 0 0 1 0 0
0.63 0 0 0 0 0 1 1 1 0 1 0 0
0.60 1 1 1 1 1 1 1 1 1 1 1 1
0.54 0 0 0 0 0 1 1 1 1 0 0 0
0.48 1 1 1 1 1 1 1 1 1 1 0 1
0.45 0 0 0 0 0 1 1 0 0 1 1 1
0.39 0 0 0 0 0 1 1 1 1 0 0 0
0.33 1 1 0 0 1 1 1 0 0 1 1 1
0.23 0 0 1 1 0 1 0 0 0 1 1 1
0.20 1 1 1 1 1 1 1 1 1 1 1 1
0.11 1 1 1 1 0 0 1 0 0 0 0 0
0.08 0 0 0 0 0 1 1 1 1 1 1 0
0.06 1 1 1 1 0 0 0 0 0 1 1 1
0.02 0 0 0 0 0 0 0 0 1 1 1 1
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III. Khai trin phn on n-butanol vi h dung mi S.2
Bng III.1. Quan st UV 254nm
Rf GL1 GL2 GL3 GL4 GS1 GS2 GS3 GS4 GS5 GI1 GI2 GY1
0.85 1 0 0 0 0 0 1 1 1 0 1 0
0.79 1 1 0 1 1 0 1 1 1 1 1 1
0.73 1 1 0 0 1 0 0 0 0 0 0 0
0.63 0 1 0 0 0 0 0 0 0 0 1 0
0.55 1 1 0 0 0 0 1 1 1 1 1 0
0.52 0 1 0 1 0 0 0 0 0 0 0 0
0.47 0 1 0 0 1 0 0 0 0 0 0 0
0.39 1 1 0 0 0 0 1 0 1 0 0 0
0.35 0 1 0 0 0 0 1 0 1 0 0 1
0.31 1 1 1 1 1 1 1 1 1 1 0 1
0.22 1 0 0 1 0 0 0 0 0 0 0 0
0.19 1 0 0 1 0 1 1 1 1 0 0 0
0.13 1 0 1 1 0 1 1 1 1 0 0 0
0.11 1 0 1 0 1 1 1 1 1 1 1 1
0.10 0 1 1 0 0 1 1 1 1 1 0 0
0.05 0 0 1 0 0 1 1 1 1 1 1 1
-
Bng III.2. Quan st UV 366nm
Rf GL1 GL2 GL3 GL4 GS1 GS2 GS3 GS4 GS5 GI1 GI2 GY1
0.86 1 0 1 1 0 0 1 1 1 1 1 0
0.80 1 1 0 1 1 0 1 1 1 0 1 0
0.71 1 1 0 0 1 0 1 1 1 0 1 0
0.64 1 0 0 0 1 0 1 1 1 0 1 0
0.56 1 1 0 1 0 0 1 1 1 1 1 1
0.52 0 0 0 0 1 0 0 0 0 0 1 0
0.40 1 1 0 0 0 0 0 0 0 0 0 0
0.38 1 1 0 0 0 0 1 1 1 1 0 0
0.33 1 1 1 0 1 0 0 1 1 1 0 1
0.31 1 1 0 0 1 0 1 0 0 0 1 0
0.24 0 0 1 0 1 0 0 0 0 0 1 1
0.18 1 0 0 0 0 0 1 1 1 1 1 1
0.12 0 0 0 0 0 0 1 0 0 0 0 0
0.07 0 0 0 0 0 0 0 1 1 0 0 1
0.04 0 0 1 0 0 0 0 0 0 1 1 0
-
Bng III.3. Hin mu bng thuc th acid sulfuric 10% v quan st nh sng
trng
Rf GL1 GL2 GL3 GL4 GS1 GS2 GS3 GS4 GS5 GI1 GI2 GY1
0.86 1 0 0 0 0 0 1 1 1 0 1 0
0.83 0 0 0 1 1 0 0 0 0 0 0 0
0.80 1 1 0 0 1 0 1 1 1 0 1 0
0.71 1 1 0 1 1 0 0 0 0 0 0 0
0.64 1 1 0 0 1 0 0 0 0 0 0 0
0.62 0 0 0 0 0 0 1 0 1 0 0 0
0.59 0 0 0 0 0 0 1 0 1 0 1 0
0.56 1 1 0 0 0 0 1 1 1 1 1 0
0.51 1 0 0 0 0 0 1 0 1 0 0 0
0.43 0 0 0 0 0 0 1 0 0 0 0 0
0.38 0 1 0 0 0 0 1 0 1 0 1 0
0.31 0 0 0 0 0 0 0 0 0 0 1 0
0.23 0 0 0 0 0 0 1 0 1 0 1 1
0.17 1 1 1 1 1 0 1 1 1 0 1 1
0.08 0 0 0 0 0 0 0 0 0 1 1 0
0.05 0 0 0 0 0 1 1 1 1 0 0 1
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IV. Khai trin dch chit ton phn vi h dung mi F.11
Bng IV.1. Quan st UV 254nm
Rf GL1 GL2 GL3 GL4 GS1 GS2 GS3 GS4 GS5 GI1 GI2 GY1
0.74 0 0 1 0 1 1 1 1 1 1 1 1
0.69 1 1 1 1 0 1 1 1 1 1 0 1
0.67 1 1 1 0 1 1 1 1 1 1 0 1
0.61 1 1 0 1 0 1 1 1 1 0 1 0
0.52 0 1 0 1 0 1 1 1 1 1 1 1
0.45 0 0 0 1 0 0 1 1 1 0 1 0
0.35 0 0 0 1 0 0 1 1 1 1 1 0
0.26 0 1 0 1 0 0 0 0 0 1 1 1
0.21 0 1 0 1 0 0 0 0 0 1 0 1
0.17 1 0 1 0 0 1 1 1 1 1 0 1
0.14 0 0 0 0 0 0 0 0 0 1 1 1
0.11 1 1 1 1 1 1 1 1 1 1 1 1
-
Bng IV.2. Quan st UV 366nm
Rf GL1 GL2 GL3 GL4 GS1 GS2 GS3 GS4 GS5 GI1 GI2 GY1
0.76 1 1 1 1 1 1 1 1 1 1 1 1
0.69 1 1 1 1 1 1 1 1 1 1 1 1
0.63 0 0 0 0 1 0 0 0 0 0 0 0
0.61 1 1 1 1 1 1 1 1 1 1 1 1
0.52 1 1 1 1 1 1 1 1 1 1 1 1
0.44 1 1 1 1 0 1 1 1 1 1 1 1
0.33 0 1 0 1 0 0 0 0 0 0 1 0
0.30 1 1 1 1 0 1 1 1 1 1 1 0
0.25 0 1 0 1 1 1 1 1 1 1 1 1
0.21 0 1 1 1 0 1 1 1 1 1 1 0
0.12 0 0 0 0 0 0 0 0 0 1 0 1
0.11 1 1 1 1 1 1 1 1 1 1 1 1
0.10 1 1 1 1 1 1 1 1 1 1 1 1
-
Bng IV.3. Hin mu bng thuc th acid sulfuric 10% v quan st nh sng
trng
Rf GL1 GL2 GL3 GL4 GS1 GS2 GS3 GS4 GS5 GI1 GI1 GY1
0.79 0 0 0 0 1 1 0 0 0 1 0 1
0.71 1 1 1 1 1 1 1 1 1 1 1 1
0.67 0 0 0 0 0 1 0 0 0 1 0 1
0.58 1 1 1 1 1 1 1 1 1 1 0 1
0.56 0 0 0 1 0 1 1 1 1 1 0 0
0.52 0 1 1 0 0 0 0 0 0 0 0 0
0.50 1 1 1 1 1 1 1 1 1 1 1 1
0.42 0 0 0 1 0 0 0 0 0 0 1 0
0.40 0 0 0 0 0 0 0 0 0 1 0 0
0.34 0 0 0 1 0 0 1 1 1 1 1 0
0.19 1 1 0 1 0 1 1 0 0 1 1 1
0.16 1 1 0 1 1 1 1 1 1 1 1 1
0.13 1 1 1 0 0 1 1 1 1 0 1 1
0.09 1 1 1 1 1 1 1 1 1 1 1 1
-
V. Khai trin phn on dch chit ethylacetat vi h dung mi F.11
Bng V.1. Quan st UV 254nm
Rf GL1 GL2 GL3 GL4 GS1 GS2 GS3 GS4 GS5 GI1 GI2 GY1
0.70 1 0 0 0 0 1 0 0 0 1 0 0
0.65 0 0 1 0 0 1 0 0 0 1 0 1
0.57 1 1 1 1 0 1 1 1 1 1 1 1
0.46 1 1 1 1 0 1 1 0 0 1 0 1
0.32 0 0 0 0 0 1 0 0 0 1 0 0
0.26 1 1 1 1 1 0 1 0 0 1 0 0
0.22 1 0 1 0 0 0 0 0 0 1 0 1
0.18 1 0 1 1 0 1 1 1 1 1 1 1
0.12 1 0 1 1 1 1 1 0 0 1 1 1
0.09 1 1 1 1 1 1 1 1 1 1 1 1
-
Bng V.2. Quan st UV 366nm
GL1 GL2 GL3 GL4 GS1 GS2 GS3 GS4 GS5 GI1 GI2 GY1
0.73 1 0 1 1 1 1 1 1 1 1 1 1
0.67 1 0 1 0 1 1 0 0 1 1 0 1
0.59 1 1 1 1 1 1 1 1 1 1 1 1
0.49 1 1 1 1 1 1 1 1 1 1 1 1
0.44 0 0 0 0 1 0 0 0 0 0 0 0
0.39 0 0 0 0 1 0 0 0 0 0 0 0
0.35 1 1 1 1 0 1 0 0 0 1 0 1
0.27 0 0 0 0 1 0 1 0 0 0 0 0
0.26 1 1 1 1 0 0 1 0 0 0 0 0
0.25 1 0 1 1 1 1 0 0 0 0 0 0
0.22 1 1 1 1 0 0 1 0 0 1 0 0
0.18 1 1 1 1 0 0 1 1 1 1 0 0
0.14 1 1 1 1 1 0 0 0 0 0 0 1
-
Bng V.3. Hin mu bng thuc th acid sulfuric 10% v quan st nh sng
trng
Rf GL1 GL2 GL3 GL4 GS1 GS2 GS3 GS4 GS5 GI1 GI2 GY1
0.79 0 0 0 0 1 0 0 0 0 0 1 0
0.74 1 1 1 1 1 1 1 1 0 1 1 1
0.64 1 0 0 0 0 0 0 0 0 1 0 1
0.56 1 1 1 1 0 1 1 1 1 1 1 1
0.53 1 1 1 1 0 1 1 1 1 0 1 0
0.47 0 1 1 1 1 1 1 1 1 1 0 1
0.41 0 0 0 0 0 1 1 1 1 0 0 0
0.36 1 0 1 0 1 1 1 1 1 1 1 1
0.30 0 0 0 0 0 1 1 1 1 1 0 0
0.21 0 0 0 0 0 0 0 0 0 1 0 0
0.18 1 1 1 1 1 1 1 0 0 1 1 0
0.15 1 1 1 1 1 1 1 1 0 1 1 1
0.11 1 0 1 1 0 0 0 0 0 1 1 1
0.09 1 0 1 1 1 1 1 1 1 1 1 1
-
VI. Khai trin phn on n-butanol vi h dung mi F.11
Bng VI.1. Quan st UV 254nm
Rf GL1 GL2 GL3 GL4 GS1 GS2 GS3 GS4 GS5 GI1 GI2 GY1
0.72 1 0 0 0 0 0 1 1 1 0 1 0
0.65 1 0 0 0 1 0 1 1 1 0
