Phân tích xác định thành phần và nhóm hoạt chất của một số dược liệu bằng phương pháp sắc ký lớp mỏng kết hợp đo mật độ quang hấp thụ TLC, DENSITOMETRY SCANNING phục vụ công tác chuẩn hoá dược liệu các sản phẩm khoa học của đ

X% : Ham lugng % atractylenolide III trong dugc lieu kho tuyet ddi m : Lugng mau thii do dugc jig kit qua trung binh ciia 2-3 lin nhic lai \: Lugng miu thii chim tren may |il V: The tich

U I O"" I

2010

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Hgp BO f^AT DO QUmo H\p THV {TLC, DEHSITOMETRY/ SCmmHG;

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B O Y T E

SAN PHAM KHOA HOC CUA DE TAI

TEN DE TAI

PHAN TICH XAC DINH THANH PHAN VA NHOM HOAT CHAT

CUA MOT SO DirOC LIEU BANG PHl/ONG PHAP SAC KY LOfP MONG

• • •

KET HOP DO MAT DO QUANG HAP THU (TLC, DENSITOMETRY/ SCANNING)

PHUC VU CONG TAC TIEU CHUAN HOA DU'OC LIEU

MUC LUC SAN PHAM KHOA HOC CUA DE TAI

TEN DE MUC

1 Cac mau dugc lieu nghien

2 Quy trinh phan tich va

nghien cuu, ky thuat TLC

THIEN MON DONG

3 Cac bai bao

cuu

bp du lieu chuan

va TLC

ve scanning

phan tich cua cdc dugc lieu

SAN PHAM KHOA HOC CUA DE TAI

Diroc lieu bach truat

Dirffc lieu chi tu*

Fructus Gardeniae

t w '< « « « >J • A * « " * *^ ^

Dirac lieu cot toai bo

Diep ha chau dang

Herba Phyllanthi amari

Du-o-c lieu dia cot bi

Du'o'c lieu kim ngan hoa

Du-o-c lieu ma d l

Folium Plantaginis

Du-o-c lieu ngu vi

tu-Fructus Schisandrae

"U

rn ^ \ kk»Mfimr

^Bt-Du-g-c lieu rau meo

Herba Orthosiphonis spiralis

Du-ac lieu thien mon dong

Radix Asparagi

ThiSn mdn dOng Thien mon ddng

Thiin moB tfSng

IWiMMSiTMUi^

SAN PHAM KHOA HOC CUA DE TAI

2 QUI TRINH PHAN TICH VA BO DlT LIEU CHUAN VE PHAN TICH CUA 10 DOI TU'ONG DtTOC Ll£U NGHIEN ClTU

KY THU A T T L C - T L C SCANNING

B A C H T R U A T {Rhizoma Atractylodis macrocephalae)

1 Nguon goc: Than re phai kho ciia cay bach truat Atractylodes macrocephala

Koidz., hg Cue {Asteraceae)

2 Thu hai, xu- ly mau: Thu hai vao thang 1 0 - 1 2 , dao lay than rl, bo rl con, co

the thai thanh lat mong, say nhe (nhiet do < 60°C) den kho

3 M o ta: Than re to (quen ggi la cu) co hinh dang thay doi, hinh chuy co nhieu

mau phinh ra, phia tren thot nho, hoac timg khiic map, nae, dai 5-10cm, duong kinh 2-5cm Mat ngoai mau nau nhat hoac xam, co nhieu mau, co van hinh hoa cue, co nhieu nep nhan dgc Chat cung, kho be gay, mat cSt khong bang, co mau vang den nau nhat, mui thorn nhe

4 Dac diem vi hoc:

• •

* Dac diem bgt dugc lieu

Bgt mau nau vang, mui tham, vi hai man Soi duai kinh hien vi thay: Mo mem gom cac te bao thanh mong, co thi mang cac te bao mo cung Te bao mo cung thanh day, thiy ro cac 6ng trao d6i Tinh thi calci oxalat hinh kim, kich thuoc khoang 0,02-0,025 mm rieng le hay tap trung thanh dam Manh mach diim, mach vach Rai rac CO sgi thanh day

- M I U ddi chilu: Atractylenolide III 0,2mg/ml methanol, lugng ch§m 5 ^1

L&p mong: TLC Silica gel 60 F254 Merck, hoat hoa a 105^C trong 1 gia truac khi

su dung

He dung moi sdc ky:

- HDM 1: n.Hexan: ethylacetat (2:1) -^ Hinh 4.1 (A,B)

- HDM 2: Ether diu: ethylacetat (50:1) -> Hinh 4.1(C)

Phdt hien:

- TT 1: DMAB - acid sulphuric,Vis / UV366nm -^ Hinh 4.1(A, B)

TT 2: Vanillin - acid sulphuric , Vis -> Hinh 4.l.C

Kitqud:mr\hAA.{A,B,C)

A t 1 2 3 4 5 A t 1 2 3 4 5

Hinh 4.1: S^c ky dd dinh tinh thanh phan nhom hoat chat trong

diro-c lieu bach truat {Rhizoma Atractylodis macrocephalae)

Ky hieu: Atractylenolide III (At); Saccharose (Sa); Cac mlu dugc lieu bach truat nghien cuu (vet 1- 5)

SKD dinh tinh terpenoid (Hinh AVIS, Hinh B/UV366, Hinh C/ VIS); SKD dinh tinh polysaccharid (Hinh D/ VIS)

Dinh tinh polysaccharid

Chudn bi mdu:

- Mau thu: 0,5g dugc lieu da xay nho, chilt vai 10 ml h6n hgp methanol, nuac

(ty le 4: 1), sieu am 30 phut, gan, lgc, dich lgc thu dugc dung dl chim sic ky Lugng cham 5 fil

Mau doi chieu: Saccharose 0,2mg/ml methanol, lugng chim 5|al

L&p mong: TLC silica gel 60 F254 Merck

He dung moi sdc ky: n.Propanol : ethylacetat: acid acetic : nuac (4:3:1:2)

- Mau thu: Can 1.0 g dugc lieu da xay nho, chiet vai 20ml methanol x 2 lan,

sieu am 30 phut moi lan Gan, gop dich chiet vao coc co mo, lam bay hai dung moi tren each thuy den can Hoa can thu dugc trong 1 ml methanol va 9ml nuac, cho vao binh lang gan, lac vai 20mlx 3 lan chloroform Gan, gop phan chloroform vao

r r r f r y

coc CO mo, hoc hai thanh can Hoa can thu dugc vua du 10ml methanol, lac deu Lgc qua giay lgc, bo 1ml dich lgc dau, lay l-2ml djch lgc tiep theo de cham sac ky

- Mau doi chieu: Dung dich atractylenolide III 0,2mg/ml trong methanol

L&p mong: HPTLC Silica gel 60 F 254 Merck, kich thuac 20 x 10 cm, hoat hoa a

105°C trong 1 gia truac khi su dung

Chdm mdu: Chim thang chuin: 2fil , 4}il x 2, 6|il , 8|j.l va lOjil; Mau thii: 4- 12^1

(chim nhic lai mlu thu 2 - 3 lin) Chim each mep kinh 15mm (chieu X), 8 mm (chilu Y), chieu rgng vet (8mm), khoang each giua cac vet (10mm)

He dung moi sdc ky: n.Hexan : ethylacetat ( 2 : 1 ) , dl bmh sic ky bao hoa dung

moi 20 phut

Phdt hien: Do TLC scanning, hap thu , k 220 nm

Cdch tiin hdnh: Kinh sau khi da trien khai vai he dung moi neu tren, cao 85mm, liy

ra, dl bay hoi hit dung moi trong h6t, thai gian 30 phiit Tiln hanh do TLC

scanning tren may CAMAG.TLC SCANNER3, buac song X 220 nm, t6c do quet

20mm/giay, chi do do remission Chuang trinh dugc thuc hien duai su diiu khiln ciia phin mim wintcats Dgc kit qua do tren may Tinh ham lugng % atractylenolide III co trong dugc lieu dugc tinh theo cong thiic sau:

m.V.100.100

X % =

u.lOOO.P(lOO-B)

X% : Ham lugng % atractylenolide III trong dugc lieu kho tuyet ddi

m : Lugng mau thii do dugc (jig) (kit qua trung binh ciia 2-3 lin nhic lai)

\): Lugng miu thii chim tren may (|il)

V: The tich dich chilt miu thii (ml)

r

P : Khoi lugng dugc lieu dem can (g)

B : Do am cua dugc lieu

Ket qua dinh liegmg:

Ket qua dinh lugng atractylenolide III trong dugc lieu bach truat dugc ghi a Bang 4.1 va Hinh 4.2

Ham lugng (%) atractylenolide HI /DL

0,0670 0,0918 0,0626 0,0500 0,0778

Do thj xac djnh khoang tuyen ti'nh

Hmh 4.2 sac ky do TLC scanning va do thj xac dinh KTT Dinh lirgng atractylenolide III trong dirg-c lieu bach truat

(Rhizoma Atractylodis macrocephalae)

C H I T U {Fructus Gardeniae)

l.Nguon goc: Qua chin phai hay say kho ciia cay danh danh Gardenia jasminoides

Ellis., hg ca phe {Rubiaceae)

2 Thu hai, xw ly mau: Thu hoach vao thang 9-11, hai lay qua chin chuyin mau

vang do, ngat bo cuong qua va loai tap, do den khi hat hoi phong len, lay ra phoi hoac say kho

3 M o ta: Qua hinh thoi ho^c hinh tning dai, dai 2-4,5cm, duang kinh l-2cm, mau

vang cam den do nau, co khi xam nau din dr xam, hai bong, c6 5-8 duang gd chay dgc qua, giiia hai gd la ranh ro ret Dinh qu^ ldm cd 5-8 la dai tdn tai, thudng bj gay cut Gdc qua hep, cdn cd vet cudng qua Vd qua mdng, gidn, hoi bong Vd qua giua mau vang due, day han Vd qua trong mau vang nga, bong, rat mdng, cd 2-3 vach ngan gia Hat nhd, mau vang cam, nau dd hoac nau den nhat, mat vd hat cd rat nhieu hat min Miii nhe Vi hai chua va d^ng

4 Dac diem vi hoc:

Dac diem bdt dugc lieu: Bdt mau vang nau hay nau dd, soi dudi kinh hien vi thiy: Sgi rieng le hay tap hgp thanh bd Manh vd qua cau tao tir nhung te bao thanh day, khoang t l bao rdng Hoac hep Be mat te bao (loai khoang rdng) thudng cd nhung Id nhd Md mem vd qua, manh ndi nhii chua cac chat du tru

- Quan sat / UV 254 ^ Hinh 4.3.A

- Anisaldehyde- acid sulphuric (TT) -^ Hinh 4.3.B

Kit qud: mnh 4.3 {A, B)

- Mau thii :lg dugc lieu da dugc tan nhd, chilt vdi 10ml methanol, sieu am 20

phiit, gan lgc liy phin dich chilt, thu dugc dung dich thii dl chim sic ky

- Miu ddi chilu: Acid chlorogenic acid 0,05 mg/ml methanol

L&p mong: TLC silica gel 60 F254 Merck, hoat hda d 105^C trong 1 gid trudc khi

sii dung

Lucmg chdm mdu: 8|il dung dich mau thii; 12(xl dung dich mau ddi chieu acid

chlorogenic

He dung moi sdc ky: Ethylacetat: acid acetic : acid formic : nudc ( 1 0 : 1 : 1 : 2 )

Phdt hien: Natural products- PEG 4000 ( TT)

Kitqud:Umh4.3.C

Dinh tinh sdc td (crocin)

Chudn bi mdu:

- Miu thii :lg dugc lieu da dugc tan nhd, chilt vdi 10ml methanol, sieu am 20

phiit, gan lgc liy phin dich chilt, thu dugc dung dich thii dl dinh tinh

L&p mong: TLC silica gel 60 F254 Merck, hoat hda d 105°C trong 1 gid trudc khi

sii dung

Lucmg chdm mdu: 6|il dung dich miu thu

He dung moi sdc ky: Ethylacetat: isopropanol: nudc (6,5 : 2,5 : 1)

Ketqud:mnh4.3.D

6.Dmh lu'o'ng:

Phuong phdp

Chudn bi mdu:

- Mau thii: Can 1,0 g dugc lieu da xay nhd, chilt vdi 25 ml methanol (x 3

lin), sieu am 30 phiit mdi lin Gan va gdp phin djch chilt va them methanol vua

dii 100ml Pha loang tiip vdi methanol dl thu dugc dung djch cd ty le lg dugc lieu

trong 500ml dung mdi Lgc qua giiy lgc, bd 2ml dich lgc diu, liy 2 - 3 ml dich lgc

tiep theo de cham sic ky

- Mau ddi chilu: Dung dich geniposide 0,20 mg/ml trong methanol

L&p mong: HPTLC Silica gel 60 F254 Merck, kich thudc kinh 20 x 10 cm, hoat

hda d 105°C trong 1 gid trudc khi sii dung

Chdm mdu: Chim thang chuin: 2^1 , 4\x\ x 2, 6}il , S\x\ va 12|il; Miu thii: 4- 10|il

(cham nhac lai miu thii 2 -3 lin) Chim each mep kinh 15 mm (chilu X), 8 mm

> ^ f r

(chieu Y), chieu rdng vet (8mm), khoang each giua cac vet (10mm)

He dung mdi sac ky: Ethylacetat: isopropanol: nudc ( 6,5 : 2,5 : 1) de binh sac ky

bao hoa dung mdi 30 phiit

Phdt hien: Do TLC scanning, hap thu A, 235nm

Cdch tien hanh: Kinh sau khi da trien khai vdi he dung mdi neu tren, cao 85mm, lay

ra, de bay hoi het dung mdi trong hdt, thdi gian 30 phiit Tien hanh do TLC

scanning tren may CAMAG.TLC SCANNER3, budc sdng A, 235 nm, tdc do quet

20mm/giay, che do do remission Chuang trinh dugc thuc hien dudi su dieu khien

ciia phan mem wintcats Dgc ket qua do tren may Tinh ham lugng % geniposide cd

trong dugc lieu dugc tinh theo cdng thiic sau :

m.V.100.100

X % =

u.lOOO.P(lOO-B) Trong dd :

X% : Ham lugng % geniposide trong dugc lieu khd tuyet ddi

m : Lugng miu thu do dugc (fig) (kit qua trung binh ciia 2-3 lin nhic lai)

u : Lugng miu thii chim tren may (|LI1)

V: Thi tich dich chilt miu thii (ml)

P : Khdi lugng dugc lieu dem can (g)

B : Do im ciia dugc lieu

Kit qua Mnh lucmg:

Kit qua dinh lugng geniposide trong dugc lieu chi tii dugc ghi d Bang 4.2, va Hinh

4.4

Bang 4.2 Ket qua dinh lugng geniposide trong dugc lieu chi tii

Ham luptg % geniposide

6,0600 6,4100 5,2800

Ghi chil

Mau thu hai

nt

nt Ham lugng geniposide trong dugc lieu chi tii khd tuyet ddi nam trong khoang 5,2800-6,4100%

Geniposide

B

— - r / ; ; « r ^ ^ /

ii :'K «kj ("Vj""

Duoc lieu chi tu

D6 thi xac dinh khoang tuyen tinh

Hinh 4.4 S^c ky do TLC scanning va do thi xac dinh khoang tuyin tmh

Dinh lirong geniposide trong dirge lieu chi tn

{Fructus Gardeniae)

C O T T O A l B O {Rhizoma drynariae)

1 Nguon goc: Than re phoi hay say khd ciia cay Cdt toai bd cdn ggi la Tac ke da

Drynariafortunei J.Sm., hg Duong xi {Polypodiaceae)

2 Thu hai va xfr ly mau: Thu hoach quanh nam, liy than rl, bd tap chit, cit bd

re con va phan la cdn sdt lai, nia sach, chgn lay cac than re to dat yeu cau, cat thanh timg manh, timg doan theo kich thudc quy dinh rdi phoi hoac siy khd

3 M o ta: Dugc lieu la doan than re, det, cong queo, nhieu phan nhanh, dai 5-15

7 A r r

cm, rdng l-3cm, day khoang 3mm, phii ldng dang vay mau nau den nau tdi Mat tren va hai ben than cd cac vet seo trdn ciia gdc la ldi hoac ldm, it khi cdn re hinh sgi The nhe, gidn, de be gay, mat gay mau nau dd, cd nhihig ddm vang xep thanh mdt vdng Miii nhe, vi nhat va hai se

4 Dac diem vi hoc:

* Dac diem bdt dugc lieu: Bgt mau vang nau, soi dudi kinh hien vi thay: Nhung manh bieu bi mau vang sim, manh md mem thanh mdng, mach gd hinh thang

5 Dinh tinh:

Dinh tinh flavonoid

Chudn bi mdu:

- M I U thu: lg dugc lieu da xay nhd, chilt vdi 10 ml methanol, sieu am 30

phiit, gan, lgc, dich lgc thu dugc diing dl chim sic ky Lugng chim 4 -6 |il

- Miu ddi chilu: Naringin 0,1 mg/ml methanol, lugng chim lO^il

L&p mong: TLC silica gel 60 F254 Merck, hoat hda d 105°C trong 1 gid trudc khi

sii dung

He dung moi sdc ky: Ethylacetat: methanol: aceton : nudc ( 1 0 : 1 : 1 : 1 )

Phdt hien: TT 1: nhdm chlorid 2% trong ethanol + UV366nm -^ Hinh 4.5 A

- TT 2: Vanillin- acid sulphuric -> Hinh 4.5.B

Kit qud:Y[mh 4.5.{A,B)

Dinh tinh hffp chdt phenolic

Chudn bi mdu:

Miu thu: lg dugc lieu da xay nhd, chilt vdi 10 ml methanol, sieu am 30 phiit, gan, lgc, dich lgc thu dugc dung dl chim sic ky Lugng chim 4 -6 ^il

Miu ddi chilu: Naringin 0,1 mg/ml methanol, lugng chim lOjil

L&p mong: TLC silica gel 60 F254 Merck, hoat hda d 105^C trong 1 gid trudc khi

sii dung

He dung moi sdc ky: Ethylacetat :acid acetic: acid formic : nudc (10:1:1:2)

Phdt hien: TT fast blue salt

Kit qud: Hinh 4.5.C

Ky hieu: Naringin (Na); Cac miu cot toai bd nghien ciiu (vlt 1- 4)

SKD djnh tinh flavonoid va hgp chit phenolic (Hinh A/UV366, Hinh B/VIS va Hinh C/VIS)

6 Dinh lirgng:

Phuong phdp

Chudn bi mdu:

- Mau thii: lg dugc lieu da dugc xay nhd, chilt vdi 15ml hon hgp methanol va

nudc( 4:1 ), sieu am 20 phiit, lam nhic lai nhu vay 3 lin Gdp dich chilt, them dung mdi viia dii 50 ml, lgc qua giiy lgc, bd 1-2 ml dich lgc diu, liy 2-3ml dich tiip theo

• » r / •

de cham sac ky

- Mau ddi chieu: Dung dich naringin 0,1 mg/ml trong methanol

L&p mong: HPTLC Silica gel 60 F254, Merck, kich thudc kinh 20 x 10 cm, hoat

hda d 105^C trong 1 gid trudc khi sii dung

Chdm mdu: Cham thang chuan: 0,3fil, 0,6)il x 2, 0,9)j,l, 1,2^1 va l,5|j.l; Miu thii:

8-14^1 (chim nhac lai miu thu 2 -3 lan) Cham each mep kinh 15 mm (chieu X), 8

mm (chieu Y), chieu rdng vet (8mm), khoang each giua cac vet (10mm)

He dung moi sdc ky: Toluen: ethylacetat: acid formic: nudc (1:12:2,5:3)

Phdt hien: Do TLC-scanning, huynh quang, X 366nm va kinh lgc K 400

Cdch tiin hdnh: Kinh sau khi triin khai sic ky, liy ra dl khd 30 phiit, nhiing dung

dich nhdm chlorid 2% trong ethanol, dl khd 30 phiit, nhiing tiip dung dich PEG

400 10% trong ethanol, dl khd tiip 30 phiit Tiln hanh do TLC scanning tren may

CAMAG.TLC SCANNER3, huynh quang, budc sdng X 366 nm, kinh lgc K 400,

tdc do quet 20mm/giay, ehi do do remission Chuang trinh duge thuc hien dudi su dieu khien ciia phan mem wintcats Dgc kit qua do tren may Tinh ham lugng % naringin cd trong dugc lieu cdt toai bd dugc tinh theo cdng thiic sau:

m.V.100.100

X% =

u.lOOO P ( I O O - B ) Trong dd :

X% : Ham lugng % naringin trong dugc lieu khd tuyet ddi

m : Lugng miu thu do dugc ()ig) (kit qua trung binh cua 2-3 lin nhic lai)

1): Lugng miu thii cham tren may {]i\)

V: The tich dich chiet miu thii (ml)

P : Khdi lugng dugc lieu dem can (g)

B : Do am cua dugc lieu

Ham lucmg % naringin

0,0567 0,2046 0,5813 0,8300

Hinh 4.6 Sac ky do TLC scanning va do thi xac dinh KTT

Dinh lirgng naringin trong dirge lieu cot toai bo

{Rhizoma Drynariae)

D I E P H A C H A U D A N G {Herba phyllanthi amari)

1 Nguon goc: Toan cay da phoi hay say khd cua cay diep ha chau Phyllanthus

amarus Lscham Et Thonn., hg thiu diu Euphorbiaceae

2 Thu hai va xur ly mau: Thu hai toan cay vao mua he, hoac mua thu, nia sach

thai ngin, dl khd (cd hiit im) d nhiet dg phdng 25 - 28° C

3 M o ta: Cay thao cao tu 30- 80 cm, than trdn mau xanh luc nhat, nhin La mgc so

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,

gan lgc liy phin dich chilt dk chim 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

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

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

/:eV^Ma:Hmh4.7.D

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

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

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

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

Kit qua:

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

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 0,36

0,34-55,56

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%

Hmh 4.8 S5c ky do TLC scanning va do thi xac dinh KTT Dmh lirgng phyllanthin trong dirge lieu diep ha chau dang

{Herba Phyllanthi amari)

D I A C O T B I {Cortex Lycii)

1 Nguon goc: Vd rl phai hay siy khd ciia cay cau ky Lycium chinense Mill., hg ca

{Solanaceae)

2 Thu hai va xvt ly mau: Thu hoach vao diu xuan va cudi thu, dao liy re, nia

sach, bdc lay vd, phai hoac siy khd

3 Mo ta: Dugc lieu cudn trdn hinh dng nhd hoac hinh mdng Mat ngoai mau vang

xam din vang nau, su si, vdi nhung dudng van mit dgc, khdng diu, dl bdc; mat trong miu vang nhat din vang xam, tuong ddi nhin cd van dgc nhd Chit nhe va gidn, dl be giy, mat giy khdng phing, ldp ngoai mau vang nau, ldp trong mau trang xam Miii nhe, vi hai nggt sau ding

4 Dac diem vi hoc:

* Dac diim vi phiu: Mat cit ngang dugc lieu la dai hinh cung Dudi kinh hiin vi nhin tii ngoai vao trong thiy: Ldp bin gdm 6-7 hang tl bao xip ldn xdn Md mim

vd la nhung te bao thanh mdng hinh da giac xip ldn xdn Bd libe cip hai chiim khoang 2/3 vi phiu cd hinh bd thap Tia rugt gdm 2-3 hang tl bao Cac tl bao chiia day tinh the calci oxalat dang cat Tang phat sinh libe-gd gdm 2-3 hang tl bao diu dan

* Dac diem bdt duge lieu: Bdt vd re dia cdt bi mau vang nau, miii nhe, vi dac biet

5 Dinh tinh:

Dinh tinh coumarin

Chudn bi mdu:

- Miu thii: 2g dugc lieu da cit nhd, chilt vdi 10ml methanol, sieu am 30 phiit,

gan, lgc liy phin dich chilt, dung dl chim sic ky Lugng chim 8-16 ^il

Miu ddi chilu: Dung dich scopoletin 0,04mg/ml trong methanol Lugng chim 6nl

L&p mong: TLC silica gel 60 F254 Merck, hoat hda ban mdng d 105°C trong 1 gid

trudc khi su dung

He dung moi sdc ky: Ethylacetat: methanol: nudc (10: 1:1)

Phdt Men: - Quan sat UV 366nm -^ Hinh 4.9.A

- Dung dich KOH/ethanol, UV366nm-^ Hinh 4.9 B

Kit qud:ms)h 4.9 {A,W)

Ky hieu: Scopoletin (Sc); Betain (Be); (3 sitosterol (Si); Cac mau dugc lieu dia cdt

bi thu hai (vet 1- 5); Huang gia bi (vet 6)

SKD dinh tinh coumarin (Hinh A/UV366, Hinh B/UV366); SKD djnh tinh

alcaloid (Hinh.C/VIS); SKD dinh tinh terpenoid (Hinh D/ VIS); SKD TLC

scarming / scopoletin va DL dia cdt bi, huynh quang X 366nm / K 540(Hinh Alva

A2)

Dinh tinh alcaloid

Chudn bi mdu:

- Mau thu: 2g dugc lieu da duge cit nhd, chilt vdi 30ml hdn hgp methanol va

nude (4:1), sieu am 30 phiit, tiip tuc dun hdi luu tren each thuy 30 phiit Dl ngudi,

gan lay phan dich chilt, lam bay hoi dung mdi din can Hda cin thu dugc trong 5ml

methanol cd chiia 0,5% acid hydrochloric, lgc qua giiy lgc, bd 1ml dich lgc diu, liy

3ml dich tiep theo dung dl chim sic ky Lugng chim 10 ul

Mau ddi chieu: Dung dich betain 4 mg/ml trong methanol Lugng chim 5^1

L&p mong: TLC silica gel 60 F254 Merck, ban nhira

He dung moi sdc ky: n.Butanol : acid hydrochlorid : nudc (8:2:1)

Phdt Men: Dragendoff cd acid h\'drochlorid

Kit qud: Hinh 4.9.C

Dinh tinh terpenoid

Chudn bi mdu:

- Mau thu: 2g dugc lieu da cit nhd, chilt vdi 10ml methanol, sieu am 30 phiit,

gan, lgc liy phin dieh chilt, dung dl chim sic ky Lugng chim 8-16 ^il

Mau ddi chilu: Dung dich p sitosterol 0,lmg/ ml Lugng chim 5^1

L&p mong: TLC silica gel 60 F254 Merck, hoat hda ban mdng d 105V trong 1 gid

He dung moi sdc ky: n.Hexan : ethylacetat (2:1)

Phdt Men: Liebermann- Burchard (TT), quan sat / VIS

^eV^M^:mnh4.9.D

6 D|nh lirgng:

Phuffng phdp

Chudn bi mdu:

- Miu thii: Can 1 g dugc lieu da dugc xay nhd, them 20 ml methanol, sieu am

30 phiit, tiep tuc chilt ndng tren each thiiy d 60°C trong 30 phiit x 2 lin Gdp dich

chiet, cd tren each thuy den can Hda can thu dugc trong vua dii 5ml methanol, lgc

bd 1ml dich lgc dau, lay 2ml dich lgc tiep theo de cham sac ky

- Mau ddi chieu: Dung dich p sitosterol 0,1 mg/ml trong methanol

L&p mong: HPTLC Silica gel 60 F254 Merck, kich thudc kinh 20 x 10 cm, hoat hda

ban mdng d 105°C trong 1 gid trudc khi sii dung

Chdm mdu: Cham thang chuan: 2\A , 4fil x 2, 6p.l, 8|LI1, va 10|j.l; mau thii 4 6|il

(cham nhac lai mau thii 2 - 3 lan) Cham each mep kinh 15 mm (chieu X), 8 mm

(chieu Y), chilu rdng vlt (8mm), khoang each gifl-a cac vet (10mm)

He dung moi sdc ky: n.Hexan : ethylacetat (2: 1), de bao hoa dung mdi sac ky 20

phiit

F

Phdt Men: Do TLC scanning, hap thu, A, 570nm

Cdch tiin hdnh: Kinh sau khi da trien khai vdi he dung mdi neu tren, cao 85mm, liy

sulphuric 10%/ ethanol (VA^), dl khd, siy d 1 0 5 ^ trong 10 phiit Tiln hanh do

TLC scanning tren may CAMAG.TLC SCANNER3, budc sdng X 570 nm, tdc do

quet 20mm/giay, chi do do remission, thdi gian do trong khoang 15- 30 phiit sau

khi say kinh Chuang trinh dugc thuc hien dudi sir diiu khiln ciia phin mim

wintcats Dgc kit qua do tren may Tinh ham lugng % p sitosterol cd trong dugc

lieu dugc tinh theo cdng thiic sau:

m.V.100.100

X% =

u.lOOO P ( 1 0 0 - B ) Trong dd :

X% : Ham lugng % hoat chit trong dugc lieu khd tuyet ddi

m : Lugng miu thii do dugc (|ug) (kit qua trung binh ciia 2-3 lin nhic lai)

u : Lugng miu thii chim tren may {\i\)

V: Thi tich djch chilt miu thii (ml)

P : Khdi lugng dugc lieu dem can (g)

B : Do am ciia dugc lieu

Kit qud:

Kit qua dinh lugng p sitosterol trong dugc lieu dia cdt bi dugc ghi d Bang 4.5 va

Hinh 4.10

F ^

Bang 4.5 Ket qua dinh lugng P- sitosterol trong dugc lieu dia cdt bi miu thu hai va

mau mua tren thi trudng

Hdm luong % fi- sitosterol

0,0205 0,0270 0,0368 0,0306 0,0245

0,0205-0,0368%

Do thi xac dinh khoang tuyen tinh

Hinh 4.10 Sac ky do TLC scanning va do thi xac dinh KTT Dinh lirong B- sitosterol trong du'o'c lieu dia cot bi

{Cortex Lycii)