le, xep thanh hai day trdng nhu la kep hinh ldng chim. Phien la hinh bau due hep, cudng la ngan, mep la nhan.
4. Dac diem vi hoc:
* Dac diem vi phau: Mat cat than trdn. Tir ngoai vao trong cd ldp bieu bi cau tao tii mdt hang te bao nhd xep deu dan. Md mem gdm cac te bao ldn trdn hoac da giac. Bd sgi cellulose d than non va lignan hoa nhieu d than gia. Vdng gd mdng cau tao tur cac mach gd gdm 5-7 te bao. Md mem rugt gdm cac te bao to trdn, da giac thanh mdng.
* Dac diim bdt dugc lieu: Bdt mau xam, vj dang nhe. Soi dudi kinh hien vi thay manh phien la mang bieu bi va md giau. Nhflng manh vd hat, manh bieu bi than, cac bd sgi kem manh mach, manh biiu bi la mang Id khi, bd sgi. Nhung manh mach soi dudi kinh hiin vi cd do phdng dai ldn cd the thay be mat vd hat mang cac cham nhd.
5. Dinh tinh:
Dinh tinh lignan Chudn bi mdu:
- Miu thii: 0,5g duge lieu da dugc tan nhd, chilt vdi 10ml methanol, sieu am 30 phiit, gan lgc liy phin dich chilt dl cham sic ky.
- Miu ddi chilu: Dung dich phyllanthin 0,05mg/ml trong methanol.
L&p mong: TLC silica gel 60 F254 Merck, hoat hda ldp mdng d 105°C trong 1 gid
trudc khi sii dung.
Lucmg chdm: 5 ^il miu ddi chilu va lO^il miu thii. He dung moi sdc ky: n.Hexan: ethylacetat (2:1).
Phdt hien: Vanillin - acid sulphuric (VS), siy d 105 °C din xuit hien vlt. Kit qua: Hmh 4.7.A.
Dinh tinh alcaloid Chudn bi mdu:
- Miu thii:
+ 0,5g dugc lieu da dugc tan nhd, chilt vdi 10ml methanol, sieu am 30 phiit,
L&p mong: TLC silica gel 60 F254 Merck,. Hoat hda ldp mdng d 105°C trong 1 gid
trudc khi sii dung.
Luang chdm: 5 |il miu ddi chilu va 10|il miu thu. He dung moi sdc ky: Toluen : ethylacetat (95 : 5). Phdt hien:: Dragendoff
i^eV^Ma:Hinh4.7.B.
Dinh tinh hap chdt phenolic
Chudn bi mdu:
Mau thii: 0,5g dugc lieu da dugc tan nhd, chilt vdi 10ml methanol 80%, sieu am 30 phiit, gan lgc lay phan dich chiet dl chim sic ky.
- Mau ddi chieu: Rutin 0,5mg/ml methanol.
L&p mong: TLC silica gel 60 F254 Merck.
F K^ F r tv
Lucmg chdm: 5 |j.l mau ddi chieu va lOfil mau thii.
He dung moi sdc ky: HDM 3: Ethylacetat: acid acetic : acid formic : nudc (10:1:1:2) Phdt hien: Fast blue salt
Kit qud: Hinh4.7.C.
Dinh tinh flavonoid
Chudn bi mdu:
- Miu thii: 0,5g duge lieu da dugc tan nhd, chiet vdi 10ml methanol 80%, sieu
am 30 phiit, gan lgc liy phin dich chiet dl cham sac ky. - Miu ddi chilu: Rutin 0,5mg/ml methanol.
L&p mong: TLC silica gel 60 F254 Merck.
F fv F F ' ^ •• <>
Luang chdm: 5 yX mau ddi chieu va 10(il mau thu.
He dung moi sdc ky: Ethylacetat: acid acetic : acid formic : nudc (10:1:1:2). Phdt hien: Natural products (NP/ PEG), UV366nm.
P 1 2 3 4 5 1 2 3 4 5
Ru 1 2 3 4 5 6 7 Ru 1 2 3 4 5 6 7
C1 D1 02
A 1 J-
Hinh 4.7. Sic ky do dmh tinh thanh phan nhom hoat chit dirge lieu
diep ha chau dang {Herba Phyllanthi amari)
Ky hieu: Rutin (Rt); Phyllanthin (P); Cac mau diep ha chau dang nghien
F
cuu (vet 1-5).
SKD dinh tinh nhdm lignan (Hinh. A/ VIS), alcaloid (Hinh. B/VIS), hgp chit phenonie (Hinh. C/VIS), flavonoid (Hinh. D/UV366)
SKD TLC scanning hgp chit phenonie/ diep ha chau dang, A, 520nm (Hinh. CI); SKD TLC scarming rutin va diep ha chau dang , huynh
6. Dinh liro-ng:
Phuffng phdp
Chudn bi mdu:
Miu thii: Can 0,5 g duge lieu da xay nhd, chilt vdi 20ml methanol (x 2 lin), sieu am 30 phiit mdi lin. Gan, gdp dich chilt, them dung mdi vd 50ml, lgc, bd 1ml dich diu, liy 2-3ml dung dich tiip theo dl chim sic ky.
1^ F F
- Mau ddi chieu: Dung dich phyllanthin 0,05mg/ml trong methanol.
L&p mong: TLC plate silica gel 60 F254 Merck, kich thudc kinh 20 x 10 cm, hoat
hda ldp mdng d 105°C trong 1 gid trude khi sii dung.
Chdm mdu: Chim thang chuin: 2|il , 4|il x 2, 8^1, 12|il va 16^1; Miu thu: 4- 6^1,
chim 2-3 lin nhic lai. Chim each mep kinh 15 mm (chilu X), 8 mm (chilu Y), chieu rdng vet (8nim), khoang each giu"a cac vet (10mm).
He dung moi sdc ky: n.Hexan : ethylacetat ( 2 : 1 ) de binh sac ky bao hoa dung
mdi 20 phiit.
Phdt Men: Tao dan xuat hoa vdi dung dich acid sulphuric 10% trong ethanol, do
F
TLC scanning, hap thu , X 61 Onm.
F I F F F
Cdch tien hdnh: Kinh sau khi da trien khai sac ky den 85mm dugc lay ra, lam khd
30 phiit, nhiing thudc thu acid sulphuric 10% trong ethanol, de khd 30 phiit, say d 105°C trong 20 phiit. Tiln hanh do TLC scanning tren may CAMAG.TLC. SCANNER3, budc sdng ?t 610 nm, tdc do quet 20mm/giay, che do do remission. Chuang trinh dugc thuc hien dudi sir dieu khien ciia phan mem wintcats. Dgc ket qua do tren may. Tinh ham lugng % phyllanthin va % lignan tdng sd (theo phyllanthin) cd trong dugc lieu dugc tinh theo edng thiic sau:
m.V.100.100 X % =
v.lOOO.P(lOO-B) Trong dd :
X% : Ham lugng % hoat chit trong dugc lieu khd tuyet ddi
m : Lugng miu thii do dugc (^g) (kit qua trung binh ciia 2-3 lin nhic lai)
1): Lugng miu thii chim tren may {\i\)
V: Thi tich dich chilt miu thu (ml) P : Khdi lugng dugc lieu dem can (g) B : Dd im ciia dugc lieu
F
Ket qua dinh lugng phyllanthin va lignan trong dugc lieu diep ha chau ding duge ghi d Bang 4.4 va Hinh 4.8.
Bang 4.4. Ket qua dinh lugng phyllanthin va lignan trong dugc lieu diep ha chau
dang TT 1. 2. 3. 4. 5. 6. Ky hieu mau DHCD 201 DHCD 202 DHCD 203 DHCD 204 DHCD 205 DHCD 206 Ham lug^g % phyllanthin (Ch&tl) 0,7175 0,4439 0,6650 0,6391 0,4345 0,6101 Ham lugrng lignan tong so 2,8049 1,7407 2,028 2,2214 0,9541 1,6747 Ty le% cdc thdnh phdn lignan Chdt I Rf (0,22-0,23) Phyllanthin 25,58 25,50 32,79 28,77 45,54 36,43 Chdt 2 Rf0,29- 0,30 (Hypophyllan thin) 18,86 15,84 15,88 14,90 12,84 14,47 Chdt 3 Rf 0,34- 0,36 55,56 58,66 51,32 56,33 41,62 49,35 Ham lugng phyllanthin trong dugc lieu diep ha ehau dang khd tuyet ddi nam trong khoang 0,4345-0,7175%.
,.A-,..-
"^V, IT, ; ^ TT, nS ^, TT 7Z TTT
Phyllanthin
B
T S — T z — ; i r
Duofc lieu diep ha chau dang
V - - •" t 4 * i * » . « « e : * i i - » Ii
. y ^
./
• 19 « n I M I ' S < I H S M ATS S M
D6 thi xac dinh khoang tuyen tinh
Hmh 4.8. S5c ky do TLC scanning va do thi xac dinh KTT Dmh lirgng phyllanthin trong dirge lieu diep ha chau dang
D I A C O T B I {Cortex Lycii)