B Y T

TRNG I HC DC H NI

NGUYN TH KIM CHI

NGHIN CU PHN LP MT S

HP CHT V C TNH CA LOI

DY THA CANH L TO (GYMNEMA

LATIFOLIUM WALL. EX WIGHT) THU

HI HO BNH

KHA LUN TT NGHIP DC S

H NI - 2014

B Y T

TRNG I HC DC H NI

NGUYN TH KIM CHI

NGHIN CU PHN LP MT S

HP CHT V C TNH CA LOI

DY THA CANH L TO (GYMNEMA

LATIFOLIUM WALL. EX WIGHT) THU

HI HO BNH

KHA LUN TT NGHIP DC S

Ngi hng dn:

ThS. Phm H Thanh Tng

DS. on Th i Ngha

Ni thc hin:

B mn Thc Vt Trng i hc Dc H Ni

B mn Dc l Trng i hc Dc H Ni

H NI 2014

LI CM N

Em xin c by t lng knh trng v li cm n su sc nht ti:

ThS Phm H Thanh Tng - B mn Thc vt, Trng i hc

Dc H Ni v DS on Th i Ngha - B mn Dc liu - Dc c

truyn, Trng i hc Y Dc Hu. Thy c trc tip hng dn, tn

tnh dy bo v gip em trong sut qu trnh hc tp, nghin cu khoa hc

v hon thnh c kha lun tt nghip ny.

Em xin gi li cm n chn thnh ti TS Nguyt Qu v ThS

Nguyn Thu Hng - B mn dc l, Trng i hc Dc H Ni

hng dn v gip em trong qu trnh thc hin kho lun.

Em xin gi li cm n chn thnh ti cc thy c, cc ch k thut vin

B mn Thc vt gip , to mi iu kin thun li em c th hon

thnh tt qu trnh lm thc nghim ti b mn.

Li sau cng, em xin c by t lng bit n su sc ti b m, cc anh

ch v bn b lun bn cnh ng h, ng vin em trong sut qu hc

tp, nghin cu v hon thnh ti ny.

Em xin chn thnh cm n!

H Ni, thng 5 nm 2014

Sinh vin

Nguyn Th Kim Chi

MC LC

DANH MC CC K HIU V CH VIT TT

DANH MC CC BNG

DANH MC CC HNH

T VN ............................................................................................... 1

CHNG 1: TNG QUAN ........................................................................ 2

1.1. LOI GYMNEMA LATIFOLIUM WALL. EX WIGHT ..................... 2

1.1.1. V thc vt ........................................................................................... 2

1.1.2. V a dng di truyn ............................................................................ 4

1.1.3. V thnh phn ho hc ......................................................................... 5

1.1.4. V tc dng sinh hc ............................................................................ 6

1.1.5. V c tnh ........................................................................................... 7

1.2. TNG QUAN V PHNG PHP NGHIN CU .......................... 8

1.2.1. V phn lp mt s hp cht ................................................................ 8

1.2.2. V nghin cu c tnh bn trng din ............................................. 9

I TNG V PHNG PHP NGHIN CU.12

2.1. NGUYN VT LIU V HA CHT, DNG C NGHIN CU .. 12

2.1.1. Nguyn vt liu ................................................................................. 12

2.1.2. ng vt th nghim ........................................................................... 12

2.1.3. Dung mi, ho cht ............................................................................ 12

2.1.4. My mc - thit b .............................................................................. 13

2.2. NI DUNG NGHIN CU ................................................................ 13

2.2.1. Nghin cu phn lp mt s cht tinh khit ........................................ 13

2.2.2. Xc nh c tnh bn trng din ...................................................... 13

2.3. PHNG PHP NGHIN CU ....................................................... 13

2.3.1. Phng php nghin cu phn lp mt s hp cht ............................ 13

2.3.2. Phng php nghin cu v c tnh bn trng din ........................ 15

2 .... KT QU NGHIN CU .................................................. 18

3.4. PHN LP MT S HP CHT ..................................................... 18

3.4.1. Qu trnh chit xut ............................................................................ 18

3.4.2. Qu trnh phn lp .............................................................................. 19

3.4.3. Xc nh cu trc cc cht phn lp c ........................................... 20

3.5. C TNH BN TRNG DIN .................................................... 24

3.5.1. nh hng ca dch chit l Dy tha canh l to n s tng trng th

trng ca chut cng trng ........................................................................... 24

3.5.2. nh hng ca dch chit l Dy tha canh l to n cc ch s huyt

hc ca chut cng trng .............................................................................. 25

3.5.3. nh hng ca dch chit l Dy tha canh l to n cc ch s sinh ho

ca chut cng trng .................................................................................... 26

3.5.4. Kt qu v m bnh hc ..................................................................... 27

3.6. BN LUN .......................................................................................... 29

3.3.1. V phng php nghin cu .............................................................. 29

3.3.2. V phn lp cc cht .......................................................................... 30

3.3.3. V c tnh bn trng din ............................................................... 32

KT LUN V XUT ........................................................................ 34

TI LIU THAM KHO

PH LC

DANH MC CC K HIU V CH VIT TT

dch chuyn ho hc

13C-NMR Ph cng hng t ht nhn cacbon 13 (Carbon-13 Nuclear

Magnetic Resonance Spectroscopy)

1H-NMR Ph cng hng t ht nhn proton (Proton Magnetic

Resonance Spectroscopy)

ALAT Alanine aminotransferase

AST Aspartat aminotrasferase

CDCl3 Cloroform

d Double nh i

DEPT Distortionless Enhancement by Polarisation Transfer

T i tho ng

EA Ethyl acetat

ITS Internal Transcribed Spacer

s Singlet nh n

SKC Sc k ct

SKLM Sc k lp mng

TMS Tetramethyl silan

DANH MC CC BNG

Bng 3.1 S liu ph NMR ca hp cht GLHE9 v cht

tham kho

21

Bng 3.2 S liu ph NMR ca hp cht GLHE7 v cht

tham kho

23

Bng 3.3 nh hng ca dch chit l Dy tha canh l to n

khi lng c th ca chut cng trng

25

Bng 3.4 nh hng ca dch chit l Dy tha canh l to n

cc ch s huyt hc ca chut cng trng

25

Bng 3.5 nh hng ca dch chit l Dy tha canh l to n

cc ch s sinh ha ca chut cng trng

26

Bng 3.6 nh hng ca dch chit l Dy th canh l to n

khi lng tim, gan, thn, lch/100g chut cng

trng

27

DANH MC CC HNH

Hnh 1.1 c im hnh thi Gymnema latifolium Wall. ex Wight

3

Hnh 1.2 on nucleotide c trng phn bit hai loi Gymnema latifolium v Gymnema sylvestre

5

Hnh 1.3 nh tnh gymnemagenin trong phn on ethylacetat trong mu Gymnema latifolium Wall. ex Wight

6

Hnh 2.1 Quy trnh th c tnh bn trng din ca dch chit Dy tha canh l to (Gymnema latifolium Wall. ex Wight)

16

Hnh 3.1 S chit xut cc phn on t Dy tha canh l to (Gymnema latifolium Wall. ex Wight)

18

Hnh 3.2 S phn lp phn on E1 t Dy tha canh l to (Gymnema latifolium Wall. ex Wight)

19

Hnh 3.3 Cu trc ha hc ca hp cht GLHE9 22

Hnh 3.4 Cu trc ha hc ca hp cht GLHE7 24

Hnh 3.5 Hnh nh vi th gan, thn l th v l chng ca chut th nghim

28

Hnh 3.6 Cc nh hng nghin cu tc dng ca lupeol

31

1T VN

Dy tha canh (Gymnema sylvestre (Retz.) R. Br. ex Schult.) c nn

y hc c truyn n Ayurveda s dng t hn 2.000 nm iu tr i

tho ng [29], [32]. Da trn kinh nghim ny, nhiu nghin cu lin quan

n Dy tha canh c thc hin v mt s sn phm t dc liu ny

c pht trin s dng trong h tr iu tr i tho ng.

Da trn nguyn l cc loi thc vt cng cp phn loi bc chi c kh

nng c cc hot cht v hot tnh sinh hc tng ng, Dy tha canh l to

(Gymnema latifolium Wall. ex Wight) thu hi huyn Cao Phong, tnh Ho

Bnh c cc nh khoa hc nghin cu tc dng h ng huyt trong

qu trnh thc hin ti nghin cu sng lc cc dc liu c tc dng iu

tr i tho ng Vit Nam nm 2010 [7]. Kt qu nghin cu cho thy

Dy tha canh l to (Gymnema latifolium Wall. ex Wight) c tc dng h

ng huyt cao hn Dy tha canh (Gymnema sylvestre (Retz.) R. Br. ex

Schult.) [7]. Do , nghin cu la chn Dy tha canh l to l mt trong

ba cy thuc c trin vng ng dng iu tr i tho ng Vit Nam.

Tuy nhin, cho n nay Vit Nam cng nh trn th gii cha c mt

cng b no v thnh phn cc cht ho hc v c tnh bn trng din ca

Dy tha canh l to. Do vy, nhm gp phn i su nghin cu v dc liu

tim nng ny, ti Nghin cu phn lp mt s hp cht v c tnh ca

loi Dy tha canh l to (Gymnema latifolium) thu hi Ho Bnh c thc

hin vi mc tiu:

Phn lp mt s hp cht trong thnh phn ho hc ca Dy tha

canh l to (Gymnema latifolium) thu hi Ho Bnh.

Xc nh c tnh bn trng din ca dch chit l Dy tha canh l

to (Gymnema latifolium) thu hi Ho Bnh.

2 TNG QUAN

1.1. LOI GYMNEMA LATIFOLIUM WALL. EX WIGHT

1.1.1. V thc vt

1.1.1.1. V tr phn loi

Theo h thng phn loi ca Takhtajan cng b nm 2009 [27], Dy tha

canh l to (Gymnema latifolium Wall. ex Wight) c v tr phn loi nh sau:

+ Ngnh Ngc Lan (Magnoliophyta)

+ Lp Ngc Lan (Magnoliopsida)

+ Phn Lp Hoa Mi (Lamiidae)

+ B Long m (Gentianales)

+ H Trc o (Apocynaceae)

+ Phn H Thin l (Asclepiadoideae)

+ Chi Gymnema R.Br.

+ Loi Gymnema latifolium Wall. ex Wight

n , Dy tha canh l to (Gymnema latifolium Wall. ex Wight) cn

c bit n vi cc tn ng ngha khc l Gymnema khandalense v

Gymnema kollimalayanum [20].

1.1.1.2. c im thc vt ca loi

Thn leo ti 6 m. Thn c l v, cc nhnh c nhiu lng t. Cung l

1,5-4 cm, c lng t dy c; phin l 8-135-8 cm, lng dy c, cng xa

trc cng dy c, gc trn, ngn nhn, gn bn 6-7 cp. Cm hoa xim dng

u thnh tng i mu, c lng dy c. Mt cm mang rt nhiu hoa, mi

thm. Cung cm hoa 1-1,5 cm, cung hoa 3-8 mm. i hnh trng, ph lng

mng. Trng hoa vng, hnh chung, nhn mt ngoi. ng hoa c 5 cp

g mang lng hng trng, cc thy hnh trng, khong 1,2 1,2 mm, ph

lng dy c hng trc, ngn hn so vi ng trng [12].

3

(b) Dng sng; (c) Cm hoa; (d) Mt trn l; (e) Mt di l; (f) Cm

hoa xim; (g) L bc ca cm hoa; (h) Mt hoa nguyn vn; (i) i; (j) Trng;

(k) Tr nh nhy; (l) Th truyn phn; (m) B nhy; (n) Qu

Hnh 1.1. c im hnh thi Gymnema latifolium Wall. ex Wight [12]

4Tr nh nhy dng hnh tr, phn ph nh dng mng ngn hn so vi

u nm nhy. Khi phn hnh thun. u nm nhy hnh nn, nh phn i.

Qu i hnh mc nhn, mi cong, 4,5-5,51,5-2 cm, c lng dy c.

Ht hnh trng thun, di khong 1,1 cm5 mm, mp dng mng; mo lng

di 3 cm [12].

Phn b: o Andaman v Nicobar, Bangladesh, Trung Quc (Qung

ng, Qung Ty, Vn Nam), n (Arunachal Pradesh, Assam, Kerala,

Maharashtra, Tamil Nadu), Myanmar, Thailand [20], Vit Nam (Cc Phng,

Ho Bnh, Thi Nguyn) [12].

1.1.2. V a dng di truyn

Nm 2012, Phm H Thanh Tng [12] cng b nghin cu xc nh

s khc bit di truyn s dng ch th phn t RAPD ca 26 mu thuc 5 loi

trong chi Gymnema R.Br., trong c 4 mu Gymnema latifolium Wall. ex

Wight. Kt qu cho thy, 3 mu min Bc Vit Nam (Ho Bnh, Thi

Nguyn v Tuyn Quang) cng nm trong cng mt nhm di truyn, trong

khi 1 mu gn khu vc min Trung Vit Nam khng cng nhm ny.

Kt qu cho thy s khc bit ca cc mu theo s phn b a l.

Nm 2013, V Th Thanh Thu [10] gii trnh t thnh cng on

gen ITS ca 8 mu trong chi Gymnema R.Br.. Kt qu xc nh s tng

ng di truyn ca 2 mu Gymnema latifolium Wall. ex Wight. l 0.99 v

pht hin on trnh t CGGCCCAAA v tr nucleotid 56 trn vng kho

st ITS1+5.8S+ITS2 hon ton khc bit v l c trng cho loi Gymnema

latifolium Wall. ex Wight so vi trnh t gen cng b trn ngn hng gen

th gii Genbank (Hnh 1.2). Kt qu chng minh kh nng nghin cu s a

dng di truyn ca cc mu s dng on trnh t gen ITS v l c s ca vic

cng b trnh t gen ca cc mu nghin cu trn ngn hng gen th gii.

5

Hnh 1.2. on nuleotide c trng phn bit hai loi Gymnema latifolium

v Gymnema sylvestre [10]

1.1.3. V thnh phn ho hc

Th nghim ban u xc nh cc nhm cht c trong Dy tha canh

l to (Gymnema latifolium Wall. ex Wight) thu hi ti Ha Bnh c thnh

phn chnh gm: saponin, flavonoid, tanin, sterol, cht bo acid amin, ng

kh v coumarin [3], [11].

Sn phm thu phn phn on ethyl acetat ca Dy tha canh l to

(Gymnema latifolium Wall. ex Wight) c pic sc k tng ng vi

gymnemagenin trn sc k (Hnh 1.3) [12].

Cho n nay, cha xc nh c ti liu no trn th gii v Vit Nam

cng b v cc hp cht c phn lp t Dy tha canh l to (Gymnema

latifolium Wall. ex Wight).

6

Hnh 1.3: nh tnh gymnemagenin trong cc phn on ethyl acetat trong

mu Gymnema latifolium Wall. ex Wight [12]

1.1.4. V tc dng sinh hc

Theo Trn Vn n, Phng Thanh Hng v cng s [7], dch chit l

Dy tha canh l to (Gymnema latifolium Wall. ex Wight) Vit Nam c tc

dng h ng huyt trn c chut bnh thng (24,07 %) tc dng cao nht

th hin sau 4 gi, ko di n 5 gi v trn chut gy tng ng huyt bi

Streptozotocin (36,31%) sau 10 ngy cho ung dch chit. Liu dng thch

hp l dy tha canh l cn ton phn c pha thnh hn dch trong nc ct

vi t l 1:1, vi liu 0,5ml/25g, tng ng vi 10g l kh/kg th trng.

Kt qu th nghim cho thy tc dng h ng huyt ca dch chit l

Dy tha canh l to (Gymnema latifolium Wall. ex Wight) mnh hn Dy tha

canh (Gymnema sylvestre (Retz) R.Br. ex Schult.). Kt qu ny c

chuyn giao cho Cng ty Dc Khoa (DK-Pharma) ca Trng i hc

Dc H Ni, l ni duy nht Vit Nam trng trt Dy tha canh l to theo

quy trnh hng dn Thc hnh tt Trng trt cy thuc (GAP- WHO).

Cng ty cho ra i sn phm Tr tiu ng DK-Betics, phn ct li l

cy thuc Dy tha canh v Dy tha canh l to.

7Ngoi cc cng b trn, hin nay trn th gii cng nh Vit Nam

cha c mt cng b chnh thc no v tc dng sinh hc ca Dy tha canh

l to (Gymnema latifolium Wall. ex Wight).

1.1.5. V c tnh

c tnh cp ca Dy tha canh (Gymnema sylvestre (Retz) R.Br. ex

Schult.) c xc nh l LD50 = 250,00 (209,21-298,75) g/kg [7].

Tham kho mt nghin cu [24] v c tnh bn trng din ca dch

chit Dy tha canh (Gymnema sylvestre (Retz) R.Br. ex Schult.) trong 52

tun vi liu t l t 0,01; 0,1; 1% cao chit vi tng lng thc n nh mc

hng ngy trn c chut c v chut ci Wista, so vi l chng m. Trng

lng c th, lng thc n tiu th c theo di hng tun cho ti 12 tun.

Sau chu k c theo di di hn, nghin cu gia k vo tun th 26 v

ln cui cng vo tun 52. ng vt th nghim c xt nghim v huyt

hc, sinh ho v bnh hc.

Kt qu cho thy, khng c chut cht trong khong thi gian 52 tun,

khng c hin tng thay i v cn nng, kh nng n ung, huyt hc, sinh

ha v bnh hc. Kt lun: khng c c tnh trn chut dng dch chit l GS

n mc liu 1% trong 52 tun.

c tnh cp ca Dy tha canh l to (Gymnema latifolium Wall. ex

Wight) c nghin cu v xc nh khng pht hin thy LD50 [7]. Vit

Nam, cha c bt c nghin cu no v c tnh bn trng din ca cc loi

trong chi Gymnema R.Br.

81.2. TNG QUAN V PHNG PHP NGHIN CU

1.2.1. V phn lp mt s hp cht

1.2.1.1. Chit xut

* Phng php ngm lnh

Sau khi chun b dc liu, dung mi c cho ngp dc liu trong

bnh chit xut, sau mt thi gian ngm nht nh (quy nh ring cho tng

loi dc liu), ti nhit d phng, rt ly dch chit (lc hoc gn) v ra

dc liu bng mt lng dung mi thch hp. tng cng hiu qu chit

xut, c th tin hnh khuy trn bng cnh khuy hoc rt dch chit di

ri li ln trn (tun hon cng bc dung mi) [5].

* Phng php ngm kit n gin

Sau khi chun b dc liu v dung mi trong bnh ngm kit. Sau mt

khong thi gian xc nh (tu tng loi dc liu), rt nh git dch chit

pha di, ng thi b sung thm dung mi pha trn bng cch cho dung

mi chy rt chm v lin tc qua lp dc liu nm yn (khng c khuy

trn). Lp dung mi trong bnh chit thng c ngp b mt dc liu

khong 3-4 cm [5].

1.2.1.2. Phn lp v xc nh cu trc ho hc

Qu trnh phn lp s dng phng php sc k ct vi cc c ch ct

hp ph v ct lc qua gel. Sc k lp mng c s dng la chn dung

mi, theo di qu trnh ra gii.

* Sc k ct hp ph:

Sc k ct hp ph da trn s phn b khc nhau ca cc cu t trong

hn hp vi hai pha khng trn ln, trong pha ng l cht lng chy qua,

pha tnh l cht hp ph dng bt mn c nhi trong ct thy tinh. Nh vy

m c th trin khai dung mi lin tc vi nhiu h dung mi khc nhau c

phn cc thay i t yu n mnh [2], [17].

9 * Sc k lc qua gel

Sc k lc qua gel l phng php phn tch cc phn t trong dung

dch da trn kch thc ca chng thng qua s trao i lp i lp li cc

phn t cht tan gia dung mi pha ng v cng dung mi c pha tnh

gi trong cc l xp ca gel. Khong kch thc l ca gel xc nh khong

kch thc phn t c phn tch qua qu trnh sc k [2], [6], [17].

* Sc k lp mng

Sc k lp mng 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 tu theo bn cht ca chng v cui cng dng li

nhng v tr khc nhau [2], [6], [17].

* Xc nh cu trc ho hc

Xc nh cu trc cc hp cht phn lp c da trn cc d liu ph:

ph hng ngoi (UV), ph t ngoi (IR), ph khi lng (MS), ph cng

hng t ht nhn (NMR) mt chiu (1H-NMR v 13C-NMR, DEPT) v hai

chiu (NOESY, COSY, HMQC, HSQC, HMBC). Khong na th k trc,

ph 1H-NMR v 13C-NMR c s dng ch yu c th xc nh cc hp

cht ho hc t dc liu. Tuy nhin, s pht trin ca ph cng hng t hai

chiu cung cp nhiu thng tin ph hn, ch ra s tng tc ln nhau gia

cc proton v carbon trong cu trc gip xc nh cu trc cc cht chnh xc

v y hn [17].

1.2.2. V nghin cu c tnh bn trng din

Nghin cu v c tnh ca thuc ng vai tr v cng quan trng

m bo tnh an ton ca thuc, thuc d tc dng mnh n my m khng

an ton th cng s khng c s dng [1].

10

c tnh bn trng din l mt trong cc ni dung v nghin cu c

tnh gm c: c tnh cp; c tnh trng din (c tnh bn cp, c tnh

c tnh bn trng din, c tnh trng din); c tnh ti ch v c tnh

chuyn bit (c tnh sinh bin chng, c tnh ung th, c tnh trn sinh sn

v pht trin) [1].

Mt s qui nh v th nghim c tnh bn trng din [1], [9]:

- Thi gian: ph thuc vo thi gian dng thuc cho ngi, thng gp

4 ln thi gian dng thuc cho ngi.

- ng vt th nghim: th, chut cng trng; ging c hay ci ty th

nghim; s lng mi l, thng vi th l 6 v vi chut l 10.

- ng dng thuc: ng dng d kin dng cho ngi, ngoi ra c

th thm mt ng dng khc.

- Liu dng: thng tnh t liu th hin tc dng trn ng vt th

nghim hoc c th ly liu dng ca ngi lm c s, ri dng php

ngoi suy tnh ra liu ca ng vt th nghim.

- Cc thi im theo di: trc khi bt u th nghim; trong khi dng

thuc, ngay sau khi dng thuc; sau khi ngng thuc mt thi gian

theo di s hi phc; xt nghim i th v vi th cc c quan th tin

hnh lc: ngay khi con vt cht, con vt cn hp hi nhng trin vng

s cht, sau khi kt thc th nghim.

- Cc thng s quan st s tu thuc ti. Mt s cc ch tiu :

o Biu hin chung: hnh vi, lng, cn nng, mc n ung

o Cc thng s huyt hc: hng cu, bch cu, tiu cu, t l

huyt sc t, thi gian mu ng, mu chy

o Cc thng s thuc chc nng gan: ALT v AST, bilirubin,

cholesterol

11

o Cc thng s thuc chc nng thn: creatinin huyt, ure

huyt

o Xt nghim i th v vi th: gan, thn, lch, tim

12

I TNG V PHNG PHP NGHIN CU

2.1. NGUYN VT LIU V HO CHT, DNG C NGHIN CU

2.1.1. Nguyn vt liu

i tng nghin cu l l ca cy Dy tha canh l to (Gymnema

latifolium Wall. ex Wight) thu hi Ha Bnh.

Cy c gim nh tn khoa hc v lu gi mu ti b mn Thc vt

Trng i hc Dc H Ni (M hiu tiu bn HNIP/18068/14).

2.1.2. ng vt th nghim

Chut cng trng chng Wistar, c 2 ging, khe mnh, cn nng t

200-220g, do Hc vin Qun y cung cp. ng vt c nui n nh trong

iu kin phng th nghim 3 ngy trc khi thc hin nghin cu.

Thc n: Thc n vin tng hp do Vin v sinh dch t trung ng cung

cp cha t l thch hp protein, carbonhydrat, m, cht khong, vitamin, cht

x.

Nc ung: Nc my sinh hot.

2.1.3. Dung mi, ho cht.

Dung mi: methanol, ethanol 70, n-butanol, n-hexan, ethyl acetat,

chloroform, aceton, nc ct... Thuc th: dung dch H2SO4 10%/Ethanol

tuyt i...

Cht nhi ct sc k: silicagel pha thng (silicagel 60; 0,040 0,063

mm (230 400 mesh ASTM); Merck), pha o (YMC, 30 - 50m, Fuji

Silysia Chemical Ltd.), Sephadex LH 20.

Bn sc k lp mng pha thng (TLC - silicagel 60 F254, 250 m,

Merck), pha o RP18 (Merck, RP C - 18 F254)....

Cc ho cht v thuc th t tiu chun phn tch theo quy nh ca

Dc in Vit Nam IV.

13

2.1.4. My mc - thit b

Cn phn tch hin s SARTORIUS TE3102S, chnh xc 0,0001 g.

bp in, t sy SHEELLAB, my c quay Buchi, bnh chit, my lc ht

chn khng, my xay dc liu ...

My o ph cng hng t ht nhn Bruker Avance AM500 FT-NMR

ti Vin Ho hc, Vin Hn lm Khoa hc v Cng ngh Vit Nam vi cht

chun ni l tetramethyl silan (TMS).

Cc dng c thy tinh: ct sc k, cc c m nhiu th tch, ng nghim,

a thy tinh, ng sinh hn, bnh gn, bnh chy sc k ...

2.2. NI DUNG NGHIN CU

2.2.1. Nghin cu phn lp mt s cht tinh khit

- Tin hnh chit xut v tch phn on cc cht theo phn cc.

- Phn lp cc cht da trn phng php sc k ct vi cc c ch ct

hp ph, ct lc qua gel. SKLM c theo di trong qu trnh ra gii.

2.2.2. Xc nh c tnh bn trng din

- Tin hnh chit xut ly dch chit ton phn ca cy Dy tha canh l

to (Gymnema latifolium Wall. ex Wight), c ly cn, phn tn trong

nc to hn dch 1:2.

- Th c tnh bn trng din vi liu nhc li trong 28 ngy trn chut

cng trng.

2.3. PHNG PHP NGHIN CU

2.3.1. Phng php nghin cu phn lp mt s hp cht

2.3.1.1. Phng php chit xut

* To dch chit ton phn

L ca cy Dy tha canh l to c ra sch, thi nh, phi, sy

nhit 50C - 60C, sau xay thnh bt th. Bt dc liu c chit bng

methanol tuyt i, phng php ngm nhit phng, thi gian 7 ngy/t

14

x 3 t, thu c dch chit ton phn. Ct thu hi dung mi methanol 50C

thu c cn ton phn.

* Chit xut cc phn on

Nguyn l: Chit xut cc cht c phn tn trong cc dung mi khc

nhau da trn phn cc v tan khc nhau ca cc cht trong cc dung

mi [17].

Tin hnh: Phn tn cn ton phn trong nc, ri tin hnh chit phn

on ln lt vi cc dung mi hu c c phn cc tng dn: n-hexan,

ethyl acetat, n-butanol. Ct thu hi dung mi di p sut gim ln lt thu

c cc cn n- hexan, ethyl acetat, n-butanol. Dch nc cn li em c cch

thu thu c cn nc.

* Chit pha rn

Nguyn l: Chit pha rn c tin hnh trn ct sc k thy tinh c

kch c khc nhau ty thuc vo lng mu em chit (thng p dng vi

lng cao chit ln trn 10g). Pha tnh hay s dng l silicagel pha thng

(silicagel 60, 0,040 - 0,063 mm (230-400 mesh ASTM), Merck). Dch chit

cao dc liu c tm vi lng va silicagel ri thc hin ct loi dung

mi thu c bt kh ti. Lng bt kh ny c a ln ct sc k, sau

ra gii bng cc h dung mi c phn cc tng dn.

Tin hnh

- Cn phn on c ha tan vo lng methanol ti thiu, sau tm

vi silicagel pha thng, t l khi lng 1:1. Khi silicagel m ny

c quay ct thu hi dung mi n th cht ti mn. Bt kh thu c

dng chit pha rn trong bc tip theo.

- Chun b ct: ct sc k c ra sch, trng bng methanol, kh t

nhin. Lt bng y ct v c nh chc chn trn gi. Cho silicagel

pha thng ln ct sao cho b dy lp silicagel khong 30 cm, hot ho

15

ct, sau a lng cht tm silicagel ni trn ln ct, g nh

bt phn b u.

- Trin khai ct vi h dung mi ra gii ln lt l n-hexan 100%, n-

hexan: ethylacetat vi cc t l 40:1, 20:1, 10:1 v ethylacetat 100%

(1,5 lt cho mi h). Hng cc phn on, c quay ct thu hi dung mi

thu c cc cn dng cao c.

2.3.1.2. Phng php phn lp

Sc k ct hp ph: Sc k ct c tin hnh trn ct silicagel pha

thng (0,040 - 0,063 mm, Merck), silicagel pha o YMC (30 - 50 m,

FuJisilisa Chemical Ltd.).

Sc k lc qua gel: s dng cht sc k l Sephadex LH-20.

Sc k lp mng: dng la chn dung mi, nh hng cc hp cht

cn phn lp v theo di qu trnh phn lp, c thc hin trn bn mng

trng sn DC - Alufolien 60G F254 (Merck), RP18 (Merck). Sc k c

quan st di nh sng n t ngoi hai bc sng 254nm v 366nm v

dng thuc th l dung dch H2SO4 10%/ethanol.

2.3.1.3. Xc nh cu trc cc hp cht phn lp c

Cht tinh khit thu c em o ph cng hng t ht nhn NMR mt

chiu (1H-NMR, 13C-NMR, DEPT) trn my Bruker Avance AM500 FT-

NMR ti Vin Ho hc, Vin Hn lm Khoa hc v Cng ngh Vit Nam vi

cht chun ni l tetramethyl silan (TMS), sau so snh vi ti liu tham

kho xc nh tn, cng thc ho hc ca hp cht.

2.3.2. Phng php nghin cu v c tnh bn trng din

2.3.2.1. Chun b cao lng

Nguyn liu: l dc liu c xay ti bt th, sy 450C ti hm m

< 5%. Dc liu c thm m bng dung mi ethanol 400 v y kn trong 2

gi.

16

Chit xut dc liu: lt mt lp bng thm nc ln trn ng thot

dch chit bt dc liu khng gy tc ng v ln vo dch chit. Sau

t giy lc ct va vn vo y bnh. Cho t t bt dc liu c

thm m vo bnh, va cho va san u v nn nh. dung mi vo bnh

chit cho ti khi c vi git chy ra th ng kha li. Tip tc dung mi

n khi cch mt dc liu 3-4cm, ngm lnh trong 48 gi. Ht thi gian

ngm lnh, m kha cho dch chit chy ra vi tc l 20 git/pht. Ch

cho thm dung mi m bo ngp mt dc liu t 2-3cm.

- Dch chit thu c em c n cn, phn tn trong nc to hn dch

1:2.

2.3.2.2. Th c tnh bn trng din.

B tr th nghim: Chut cng trng c 2 ging c chia ngu nhin

thnh 3 l, cho ung thuc th hoc nc vo mt gi nht nh lin tc trong

28 ngy, cn c vo liu tc dng trn ngi 10g dc liu kh/kg th trng:

- L chng (n=10): ung nc vi th tch 1ml/100g chut.

- L th liu 1 (n=10): ung dch chit nc cao Dy tha canh l to

vi liu 1,4 g/kg chut (tnh theo dc liu kh).

- L th liu 2: gp 3 liu 1 (n=10): ung dch chit nc cao Dy tha

canh l to vi liu 4,2 g/kg chut (tnh theo dc liu kh).

Hnh 2.1. Quy trnh th c tnh bn trng din ca dch chit

17

Dy tha canh l to (Gymnema latifolium Wall. ex Wight)

Thng s nh gi

- Tnh trng ton thn: Biu hin ca ng vt thc nghim: tnh

trng da, lng, mt, s tit dch, phn, nc tiu, hot ng t

nhin, hnh vi, s tiu th thc n, nc ung (theo di hng

ngy). S thay i khi lng c (cn hng tun).

- Huyt hc: Cc thng s: s lng hng cu, nng

hemoglobin, t l hematocrit, th tch trung bnh hng cu, lng

huyt sc t trung bnh, nng huyt sc t, s lng tiu cu, s

lng bch cu v t l % t bo lympho.

- V sinh ho: Cc thng s: Hot AST, hot ALT,

cholesterol ton phn, protein ton phn, creatinin huyt thanh.

- V m bnh hc: i th cc c quan: quan st cm quan cc c

quan tim, gan, thn, lch. Cn ngay khi lng cc c quan v tnh

t l so vi khi lng ton b c th.

- Tiu bn vi th gan, thn (ly ngu nhin 30% ng vt mi l)

X l s liu: Phng php hu kim ANOVA, s liu c trnh by

di dng M SD (M: gi tr trung bnh ca tng l, SD: lch chun).

18

KT QU NGHIN CU 3.1. PHN LP MT S HP CHT

3.1.1. Qu trnh chit xut.

Bt dc liu (5 kg) c chit xut bng phng php ngm lnh thu

c 315g cn ton phn, sau tin hnh lc phn on thu c khi lng

cn ln lt l: n-hexan: 50g, ethylacetat: 60g, butanol: 80g, nc: 75g. Cn

phn on ethylacetat c em chit pha rn thu c 5 phn on t E1

E5 (Hnh 3.1).

Hnh 3.1. S chit xut cc phn on t Dy tha canh l to

(Gymnema latifolium Wall. ex Wight)

19

3.1.2. Qu trnh phn lp

Phn on E1 c chn tip tc phn lp (Hnh 3.2).

E1 c khai trin trn sc k ct pha thng vi h dung mi n-hexan :

ethylacetat (40:1), thu c 2 phn on E1A v E1B.

Hnh 3.2. S phn lp phn on E1 t Dy tha canh l to

(Gymnema latifolium Wall. ex Wight)

Phn on E1A c tip tc phn lp trn sc k ct pha thng vi h

dung mi n-hexan : cloroform (20:1) thu c 7 phn on E1A1 E1A7.

20

Phn on E1A6 tip tc c trin khai bng phng php sc k ct

pha o vi h dung mi aceton : methanol : nc (1:1:0,2) thu c cc phn

on c k hiu E1A6A E1A6E.

Phn on E1A6E c a ln ct sc k pha thng vi h dung mi

n-hexan : cloroform (15:1) thu c E1A6E1 tng i sch. E1A6E1 c

trin khai trn sc k ct lc qua gel Sephadex LH-20, ra gii bng methanol

tuyt i, thu c 1 hp cht sch s 1 (k hiu l GLHE9).

Phn on E1A7 c trin khai trn sc k ct pha thng vi h dung

mi n-hexan : ethylacetat (10:1) thu c E1A7A. E1A7A c trin khai

trn sc k lc qua gel Sephadex LH-20, ra gii bng methanol tuyt i, thu

c 1 hp cht sch s 2 (k hiu l GLHE7).

3.1.3. Xc nh cu trc cc cht phn lp c

3.1.3.1. Hp cht 1 (GLHE9)

Hp cht 1 (k hiu l GLHE9) c tch ra di dng bt kt tinh

trng hnh kim; khi kim tra bng sc k bn mng pha thng vi dung mi

n-hexan: ethyl acetat (10:1, v/v) hin vt mu hng vi thuc th H2SO4 10%,

Rf = 0,47.

Ph 1H-NMR ca GLHE9 ch ra tn hiu singlet c trng ca 7 nhm

methyl ti H 2,13; 2,04; 0,98; 0,87 (x2); 0,85 (x2); 1 tn hiu proton ca

nhm oxymethin ti H 4,48 (dd, 10,0; 6 Hz). T gi tr ca hng s tng tc

J suy ra proton ca nhm oxymethin phi c cu hnh (chim v tr axial

trong cu dng gh ca vng A khung dammaran).

Ph 13C v DEPT ca hp cht ny ch ra tn hiu ca 24 nguyn t

cacbon bao gm 7 nhm methyl, 8 nhm methylen, 5 nhm methin v 6

cacbon bc bn. Trong , tn hiu ti C 212,3 l ca nhm ceton (C=O), ti

C 171,0 l ca nhm acetyl (-COO).

21

Bng 3.1: S liu ph NMR ca hp cht GLHE9 v cht tham kho

V tr C

Cht tham kho [16] GLHE9

Ca,b Ha,c (J, Hz) C, b DEPT Ha, c (J, Hz) 1 38,78 _ 38,8 CH2 _ 2 23,69 _ 23,7 CH2 _ 3 80,87 4,48 dd (10,0; 6,0) 80,8 CH 4,48 dd (10,0; 6,0) 4 37,90 _ 37,9 C _ 5 55,95 _ 55,9 CH _ 6 18,13 _ 18,1 CH2 _ 7 35,46 _ 35,4 CH2 _ 8 40,50 _ 40,5 C _ 9 50,61 _ 50,6 CH _ 10 37,11 _ 37,1 C _ 11 21,24 _ 21,2 CH2 _ 12 25,97 _ 25,9 CH2 _ 13 45,12 1,92 (m) 45,1 CH _ 14 50,05 _ 50,0 C _ 15 31,55 _ 31,5 CH2 _ 16 25,57 _ 25,5 CH2 _

17 54,26 2,59 ddd

(11,0; 11,0; 6,5) 54,2 CH 2,59 ddd

(11,0; 11,0; 6,0) 18 15,57 0,98 (s) 15,5 CH3 0,98 (s) 19 16,30 0,87 (s) 16,3 CH3 0,87 (s) 20 221,44 _ 212,3 C _ 21 30,00 2,13 (s) 30,0 CH3 2,13 (s) 28 27,97 0,853 (s) 27,9 CH3 0,85 (s) 29 16,50 0,849 (s) 16,5 CH3 0,85 (s) 30 15,86 0,87 (s) 15,8 CH3 0,87 (s)

OAc 21,33 2,05 (s) 21,3 CH3 2,04 (s)

171,05 _ 171,0 C _ a o trong DMSO-d6, b o trong CDCl3 , b 125 MHz, c 500 MHz, dng TMS

lm cht chun ni

Cc d kin ph cho php d on 1 l mt triterpen c bn vng, cng

thc phn t l C26H42O3. Kt hp vi ti liu [16], hp cht 1 c xc nh

l 3-acetoxy-22,23,24,25,26,27-hexanordammaran-20-on.

22

Hnh 3.3. Cu trc ho hc ca hp cht GLHE9

3.1.3.2. Hp cht 2 (GLHE7)

Hp cht 2 (k hiu l GLHE7) c tch ra di dng bt kt tinh

trng, tan tt trong chlorofom; khi kim tra bng sc k bn mng pha thng

vi dung mi aceton : nc (15:1, v/v) hin vt mu hng vin xm vi thuc

th H2SO4 10%, Rf = 0,6.

Ph 1H-NMR ca GLHE7 ch ra tn hiu singlet ca 8 nhm methyl ti

H 2,0; 1,68; 1,03; 0,93; 0,85; 0,84; 0,83; 0,78 v 1 nhm exo-metylen ti H

4,56; 4,68. Tn hiu cng hng ti H 4,44 dd (10,5; 5,0) thuc v proton ca

nhm methin gn vi oxy. Cc hng s tng tc J1 = 10,5 Hz; J2 = 5,0 Hz

tng ng vi tng tc diaxial, axial-equatorial, chng t proton ny nh

hng axial (hay cu hnh ).

Ph 13C v DEPT ca hp cht ny ch ra tn hiu ca 32 nguyn t

cacbon bao gm 8 nhm methyl, 11 nhm methylen, 6 nhm methin v 7

cacbon bc bn. Trong , tn hiu ti C 109,4 thuc v nhm exo-methylen

(C-29) v tn hiu ti C 81,0 thuc v nhm methin gn vi oxy (C-3).

23

Bng 3.2. S liu ph NMR ca hp cht GLHE7 v cht tham kho

V tr C

Cht tham kho [15] GLHE7 Ca,b Ca, b DEPT Ha, c (J, Hz)

1 37,8 37,8 CH2 _ 2 21,0 21,0 CH2 1,9 m 3 80,9 81,0 CH 4,44dd (10,5; 5,0) 4 43,0 43,0 C _ 5 55,4 55,4 CH _ 6 18,3 18,2 CH2 _ 7 37,1 37,1 CH2 _ 8 38,4 38,4 C _ 9 50,3 50,4 CH _

10 34,2 34,2 C _ 11 18,3 18,3 CH2 _ 12 23,8 23,7 CH2 _ 13 38,1 38,0 CH _ 14 42,8 42,8 C _ 15 27,5 27,5 CH2 _ 16 35,6 35,6 CH2 _ 17 40,9 40,9 C _ 18 48,3 48,3 CH _ 19 48,0 48,0 CH _ 20 150,8 151,0 C _ 21 29,9 29,9 CH2 _ 22 40,0 40,0 CH2 _ 23 25,1 25,1 CH3 0,85 (s) 24 16,6 16,5 CH3 0,83 (s) 25 16,3 16,2 CH3 0,84 (s) 26 16,1 16,1 CH3 1,03 (s) 27 14,6 14,5 CH3 0,93 (s) 28 18,1 18,1 CH3 0,78 (s)

29 109,3 109,4 CH2 4,68 (br.s)

4,56 (br.s) 30 19,3 19,3 CH3 1,68 (s)

OAc 170,8 171,0 C _ 21,4 21,3 CH3 2,0 (s)

a o trong CDCl3 -d, b 125 MHz, c 500 MHz dng TMS lm cht chun ni

24

Cc d kin ph cho php d on 2 l mt triterpenoid 5 vng c khung

lupan, cng thc phn t l C32H52O2. Kt hp vi ti liu [15], hp cht 2

c xc nh l lupeol acetat.

Hnh 3.4. Cu trc ho hc ca hp cht GLHE7

3.2. C TNH BN TRNG DIN

3.2.1. nh hng ca dch chit l Dy tha canh l to n s tng trng

th trng ca chut cng trng

Trong qu trnh nghin cu, chut cc l u n ung, hot ng bnh

thng, mt sng, lng mt, phn kh, nc tiu bnh thng. Trong sut 28

ngy nghin cu, cn nng chut cng tng u cc l (Bng 3.3). Khng

c s khc bit v khi lng chut gia l th v l chng ti tt c cc thi

im nghin cu (p>0,05).

25

Bng 3.3. nh hng ca dch chit l Dy tha canh l to n khi

lng c th ca chut cng trng

L n N0 (g) N7 (g) N14 (g) N 28 (g) Chng1 10 180,0 11,55 191,0 7,37 205,5 11,65 220,5 20,52 Liu 12 10 175,0 12,69 190,0 8,16 205,0 7,07 221,3 20,62 Liu 23 10 178,0 9,19 192,0 10,33 209,0 8,75 226,4 13,18

p1-2 = 0,51 p1-3 = 0,89

p1-2 = 0,95 p1-3 = 0,95

p1-2 = 0,99 p1-3 = 0,62

p1-2 = 0,99 p1-3 = 0,72

(S liu biu din di dng M SD)

3.2.2. nh hng ca dch chit l Dy tha canh l to n cc ch s

huyt hc ca chut cng trng

S khc bit v ch s huyt hc ti thi im trc ung mu th (N0),

sau ung mu th 14 v 28 ngy gia cc l th nghim so vi l chng

khng c ngha thng k (p> 0,05).

Bng 3.4. nh hng ca dch chit l Dy tha canh l to n cc ch

s huyt hc ca chut cng trng

n N0 N14 N28 WBC

(109/L) Chng 10 10,93 2,00 12,33 3,51 16,05 3,83 Liu 1 10 11,67 4,17 12,05 3,01 14,12 2,88 Liu 2 10 11,94 3,07 10,95 2,07 15,1 3,22

RBC (1012/L)

Chng 10 6,82 0,73 7,13 0,71 6,98 0,60 Liu 1 10 6,29 0,55 7,65 0,51 7,63 0,29*

Liu 2 10 6,20 0,76 7,65 0,55 7,44 0,76 HGB (g/dL)

Chng 10 10,91 0,88 11,60 0,98 11,93 1,09 Liu 1 10 10,31 0,71 12,18 0,89 12,79 0,87 Liu 2 10 10,23 1,09 12,27 1,11 12,51 1,65

HCT(%) Chng 10 35,01 3,14 36,57 3,39 35,28 3,40 Liu 1 10 32,44 3,17 38,99 2,72 38,46 2,40 Liu 2 10 31,65 4,57 39,30 3,56 37,89 5,31

MCV (fL) Chng 10 51,43 2,36 51,32 2,43 50,56 2,29 Liu 1 10 51,55 2,53 30,95 2,52 50,37 2,15 Liu 2 10 50,92 1,88 51,33 2,23 50,77 2,77

MCH (pg) Chng 10 16,04 0,67 16,30 0,99 17,12 1,13 Liu 1 10 15,43 3,26 15,91 0,74 16,73 0,92 Liu 2 10 16,54 0,65 16,02 0,62 16,79 0,89

26

MCHC (g/dL)

Chng 10 31,20 0,82 31,76 1,04 33,85 0,95 Liu 1 10 31,90 1,87 31,25 0,41 33,26 0,75 Liu 2 10 32,48 1,91 31,23 0,37 33,06 0,54

PLT (109/L)

Chng 10 522,6 136,31 441,30 49,38 558,0 152,9 Liu 1 10 499,4 136,4 459,1 153,6 501,2 143,1 Liu 2 10 530,6 93,34 408,9 116,5 496,3 101,4

Lympho (%)

Chng 10 69,25 6,48 70,01 7,99 72,13 16,36 Liu 1 10 73,30 14,96 70,50 3,90 78,59 22,88 Liu 2 10 80,35 13,91 72,90 11,7 81,78 12,29

(s liu biu din di dng M SD, *, p0,05) .

Bng 3.5. nh hng ca dch chit l Dy tha canh l to n cc ch

s sinh ha ca chut cng trng

N N0 N14 N28 AST (U/L) Chng 10 148,28 85,95 141,93 57,39 157,40 19,83

Liu 1 10 157,96 75,62 104,68 58,56 174,40 36,37 Liu 2 10 158,33 76,71 120,54 52,75 166,50 23,43

ALT (U/L) Chng 10 48,38 9,64 48,66 8,22 45,30 3,51 Liu 1 10 45,54 8,25 46,91 8,98 47,10 10,31

Liu 2 10 46,26 10,49 41,34 7,40 53,00 4,40 Creatinin (mol/L)

Chng 10 91,79 27,70 61,23 16,66 86,07 27,83 Liu 1 10 75,20 20,33 62,98 35,25 67,54 23,94 Liu 2 10 76,23 19,73 73,91 64,64 69,84 19,92

Cholesterol (mmol/L)

Chng 10 1,67 0,19 1,17 0,13 1,30 0,19 Liu 1 10 1,82 0,35 1,03 0,28 1,44 0,28 Liu 2 10 1,84 0,35 1,06 0,19 1,38 0,25

Protein (g/L)

Chng 10 77,20 8,19 76,73 7,15 71,59 22,04 Liu 1 10 79,77 7,68 82,86 18,18 70,12 12,36 Liu 2 10 72,75 8,51 81,76 13,09 69, 07 13,12

27

(s liu biu din di dng M SD, *, p< 0,05 khi so vi l chng)

3.2.4. Kt qu v m bnh hc.

V i th: khng thy c s thay i i th ca cc c quan tim, gan,

thn, lch gia cc l.

Bng 3.6: nh hng ca dch chit l Dy tha canh l to n khi lng

tim, gan, thn, lch/100g chut cng trng

L n Tim Gan Thn Lch

Chng1 10 0,37 0,03 3,46 0,39 0,56 0,04 0,38 0,11

Liu 12 10 0,37 0,05 3,47 0,29 0,61 0,07 0,34 0,08

Liu 23 9 0,33 0,03 3,41 0,27 0,55 0,06 0,29 0,03

(s liu biu din di dng M SD *, p0,05)

- V vi th: Tiu bn vi th gan, thn c thc hin v nh gi ti B

mn Gii phu bnh Trng i hc Y H Ni. Kt qu cho thy, sau 28

ngy ung mu th lin tc, cu trc thn trong gii hn bnh thng,

khng thy xut hin tn thng. Trn cu trc vi th gan, cu trc gan khng

b o ln, khng thy t bo gan bin i bt thng, ri rc xut hin cc t

bo gan thoi ha (khoanh trn) do s xm nhp ca cc k sinh trng (sn

l gan). Tuy nhin khng c s khc bit gia l dng thuc v l chng

(Hnh 3.5).

28

Hnh nh vi th thn l chng Hnh nh vi th gan l chng

(Nhum HEx100) (Nhum HEx100)

Vi th thn l th thuc liu 1 Vi th gan l th thuc liu 1

(Nhum HEx100) (Nhum HEx100)

Vi th thn l th thuc liu 2 Vi th gan l th thuc liu 2

(Nhum HEx100) (Nhum HEx100)

Hnh 3.5. Hnh nh vi th gan, thn l th v l chng ca

chut th nghim

29

3.3. BN LUN

3.3.1. V phng php nghin cu

Phng php ngm lnh, ngm kit n gin uc dng to dch

chit ton phn. Hai phng php ny tuy khng mang li hiu sut cao

nhng vi u im n gin, kinh t, ph hp vi mc ch nghin cu nn

c la chn.

Sc k ct (SKC) v sc k lp mng (SKLM) c s dng trong

ti cng l nhng phng php ph bin v thng quy trong nghin cu v

dc liu. SKC cho hiu qu tch cao, phn on d tinh sch, c bit l t

gy bin tnh, bo ton nguyn vn cht, tin hnh n gin. SKLM cng l

mt phng php n gin, kinh t v nhanh gn c th dng cho phn tch

nh tnh v nh lng cng nh l tinh ch hp cht t thin nhin. Trong

nghin cu, SKLM ng vai tr nh mt cng c b tr ti u ho iu kin

ca sc k ct, bao gm la chn dung mi, kim tra s tinh khit ca phn

on phn lp c.

V xc nh cht, trong s loi ph ph bin thng c s dng nh:

ph hng ngoi (UV); ph t ngoi (IR); cng hng t ht nhn (NHR) mt

chiu (1H-NMR v 13C-NMR, DEPT) v hai chiu (HMBC, COSY, HMQC,

HSQC); khi ph (MS). ti la chn v s dng loi ph chnh l cng

hng t ht nhn mt chiu (1H-NMR, 13C-NMR, DEPT) kt hp vi ti liu

tham kho c th xc nh tn cng nh cng thc ho hc ca hp cht

phn lp.

V th c tnh bn trng din: nghin cu c thit k trn m hnh

chut cng trng; ng ung, vi 2 mc liu (liu 2 gp 3 ln liu 1) v

dng trong thi gian 28 ngy da trn mt s hng dn v th nghim c

tnh ca thuc [1], [9], [23]. Do T l mt bnh mn tnh, vic dng thuc

c th ko di sut i nn cn phi c th nghim c tnh vi thi gian di

30

hn. Trong phm vi nghin cu ca ti, vic nh gi xc nh c tnh

ng ung vi liu lp li trong 28 ngy ca th nghim c thc hin sau

khi c kt qu ban u v c tnh cp, to tin cho th nghim c tnh

trong thi gian di hn, gp phn vo vic chng minh s an ton ca dc

liu [23].

3.3.2. V phn lp cc cht

Hp cht (1): 3-acetoxy-22,23,24,25,26,27-hexanordammaran-20-

on l dn cht nor-dammaran triterpenoid, cng c phn lp t mt s

cy Ficus pumila L. [16], Panax quinquefolium L. [18], Viburnum

mongolicum [30]. Mt s dn cht nh octanordammaran, trinordammaran,

hexanordammaran t Viburnum mongolicum c chng minh tc dng

gy c trn t bo ung th [30], dn cht nordammaran t Gynostemma

pentaphyllum c tc dng chng vim [31]. Tuy nhin v tc dng dc l c

th ca hp cht (1) th vn cha c bt k cng b no do c th t ra

hng nghin cu su hn v tc dng ca hp cht ny.

Hp cht (2): Lupeol acetat l dng mui ca hp cht lupeol thuc

dn cht triterpenoid 5 vng c phn lp t nhiu dc liu nh

Tournefortia hartwegiana Steud (hm lng ln ti 310mg/kg), r cau (Areca

catechu L.) [8], Phyllanthus reticulatus [14], Himtanthus drasticus [19]

Trong sut 3 thp k qua, Lupeol c cc nh khoa hc nghin cu vi

nhiu hng tc dng khc nhau. Cc nh hng tc dng c nghin

cu lin quan bao gm: (1) Khng vim, (2) iu tr bnh thn, (3) Gii c

gan, (4) Khng khun, (5) Vim khp, (6) Bnh tim mch, (7) Bnh ung th

v (8) H ng huyt [26] (Hnh 3.6)

31

Hnh 3.6: Cc nh hng nghin cu tc dng ca lupeol [26]

Mt trong nhng tc dng ng quan tm ca Lupeol lin quan ti dc

liu Dy tha canh l to l tc dng h ng huyt. Lupeol c tc dng h

ng huyt trn m hnh in vitro, in vivo v mt s tc dng lm gim nguy

c pht trin ca bnh T trn m hnh ng vt thc nghim [13], [21],

[22], [25]. C ch tc dng ca lupeol trn bnh i tho ng cho n nay

vn cha c nghin cu y tuy nhin cc nghin cu ban u cho php

d on tc dng theo cc c ch c ch protein tyrosine phosphatase 1B

(mt protein c tc ng quan trng trong vic c ch tc dng ca insulin v

gy ra T type 2 v bo ph) [21], c ch enzym alpha-amylase [13] v -

glucosidase (hai enzym lin quan n s trao i v hp thu glucose trong c

th) [22], [25].

Nh vy, vic phn lp c lupeol acetat trong Dy tha canh l to c

th d on c c ch tc dng h ng huyt ca dc liu ny vi c

ch tng t Lupeol. Do , a ra trin vng nghin cu tc dng lin quan

n h ng huyt ca Lupeol acetat phn lp t Dy tha canh l to.

32

y l cng b u tin v s c mt ca hai hp cht Lupeol acetat v

3-acetoxy-22,23,24,25,26,27-hexanordammaran-20-on t dch chit l ca

loi Dy tha canh l to (Gymnema latifolium Wall. ex Wight).

3.3.3. V c tnh bn trng din

Chc nng to mu c th hin qua s lng cc loi huyt cu, hm

lng hemoglobin, t l hematocrit, t l cc dng bch cu. Kt qu cho thy

khng c s khc bit cc thng s huyt hc ni trn ti cc thi im khc

nhau trong mi l th nghim, cng nh gia l th v l chng. iu ny

chng t dch chit Dy tha canh l to vi 2 liu th nghim khng lm thay

i chc nng to mu ca chut trong thi gian 28 ngy.

Trong c th, gan l c quan ch yu m nhn chc nng chuyn ho

mt s cht v kh c. Cc thuc sau khi ung hoc tim, phn ln u qua

gan chuyn ho. V vy c tnh ca thuc thng nh hng n gan. c

bit khi dng thuc di ngy i vi cc bnh mn tnh th kh nng tch lu

thuc trong c th cng ln, nguy c gy tn thng gan cng cao. ti

nghin cu c tnh ca thuc trn gan thng qua m bnh hc v cc xt

nghim thng quy l hot AST, ALT, nng cholesterol ton phn,

protein ton phn trong huyt thanh. AST, ALT l hai enzym xc tc cho qu

trnh chuyn ho acid amin gan. S tng hot cc enzym ny gn lin vi

s tn thng hoc suy gim chc nng gan. ASAT khng ch c mt gan

m cn c mt mt s m khc c bit l t bo c tim do vy khi ASAT

tng cao cng c th do tn thng t bo c tim. Khc vi ASAT, ALAT c

nhiu nht gan, chng ch khu tr trong bo tng ca t bo nhu m gan,

tng mnh ngay khi c s thay i tnh thm ca mng t bo gan hay nhng

tn thng hy hoi t bo gan do c coi l enzym c hiu vi gan hn

[4]. Mt khc, khi gan b suy gim chc nng, nng cholesterol ton phn

v protein ton phn trong huyt thanh thng c xu hng gim. Kt qu

33

thc nghim cho thy, mi l, hot AST, ALT, protein ton phn,

cholesterol ton phn thay i khng c s khc bit c ngha thng k gia

cc l th nghim.

Cng vi gan, thn cng l c quan thi tr c tnh quan trng ca c

th. Chc nng thn c nh gi thng qua ch tiu nng creatinin trong

huyt thanh. Bi creatinin c bi tit hu ht qua thn nn nu nng

creatinin huyt thanh tng cao c th bo hiu ri lon chc nng thn. Kt

qu nghin cu cho thy, c 2 liu th nghim trong 28 ngy u khng lm

thay i nng creatinin huyt thanh so vi ban u cng nh cng thi

im.

Kt qu tiu bn vi th gan, thn cho thy cu trc thn trong gii hn

bnh thng, khng xut hin tn thng, cu trc gan khng b o ln,

khng thy t bo gan bin i bt thng. Ri rc xut hin cc t bo gan

thoi ho do s xm nhp ca cc k sinh trng (sn l gan), tuy nhin

khng c s khc bit gia l dng thuc v l chng do khng nh hng

ti kt lun v c tnh ca dch chit.

Nh vy, dch chit l Dy tha canh l to vi liu 1,4 g/kg v 4,2 g/kg

(tnh theo dc liu kh) dng trong 28 ngy lin tc khng nh hng n

chc nng gan, thn ca chut thc nghim. Kt qu ny chng minh an

ton ca dch chit l Dy tha canh l to trong th nghim c tnh bn

trng din theo m hnh liu lp li.

Tm li, cng vi kt qu v c tnh cp c cng b [7] v kt qu

v c tnh ca ti bc u chng minh tnh an ton ca Dy tha canh

l to, iu ny rt c ngha cho vic a Dy tha canh l to (Gymnema

latifolium Wall. ex Wight) tr thnh dc liu h tr v iu tr T.

34

KT LUN V XUT

1. KT LUN

chit xut, phn lp v xc nh cu trc ho hc 2 hp cht t Dy

tha canh l to: (1) 3-acetoxy-22,23,24,25,26,27-hexanordammaran-20-on

v (2) Lupeol acetat.

Dch chit l Dy tha canh l to (Gymnema latifolium Wall. ex Wight)

vi liu 1,4 g/kg v 4,2 g/kg (tnh theo dc liu kh) cho chut cng trng

dng theo ng ung lin tc 28 ngy khng nh hng n tnh trng ton

thn, s pht trin khi lng c th, chc nng to mu, chc nng gan, thn,

i th, khi lng tim, gan, thn, lch v vi th gan thn.

2. XUT

T cc kt qu nghin cu v tc dng h ng huyt tng i mnh

v ko di, nghin cu ho hc, cng nh nghin cu c tnh cp v bn

trng din ca dch chit l Dy tha canh l to (Gymnema latifolium Wall.

ex Wight) khng thy biu hin c tnh trn cc c quan to mu, gan, thn.

iu ny cho thy trin vng Dy tha canh l to (Gymnema latifolium Wall.

ex Wight) c th l thuc dng cho bnh nhn T, do ti xin a ra

mt s khuyn ngh:

- Tip tc nghin cu chit xut, phn lp v xc nh cu trc ho

hc trong dch chit l Dy tha canh l to (Gymnema latifolium

Wall. ex Wight).

- i su tm hiu v xc nh tc dng sinh hc ca cc hp cht c

phn lp.

TI LIU THAM KHO

TING VIT

1. Trung m (2006), Nghin cu c tnh ti ch, Cc phng

php nghin cu tc dng dc l ca thuc t dc tho, Nh xut

bn Khoa hc v K thut, H Ni.

2. Nguyn Vn n, Nguyn Vit Tu (1985), Phng php nghin cu

ho hc cy thuc, Nh xut bn y hc, H Ch Minh.

3. Phm Vn Hi (2010), Nghin cu c im thc vt, thnh phn ha

hc v tc dng sinh hc ca cy Dy tha canh l to Ha Bnh,

Kho lun tt nghip dc s. Th vin i hc Dc H Ni.

4. Hong Th Kim Huyn (2007), Dc lm sng, NXB B y t.

5. T Minh Kong (2007), K thut sn xut dc phm, Nh xut bn y

hc, H Ni, tr. 157 - 158.

6. B mn Dc liu (2006), Phng php nghin cu dc liu, Th

vin trng i hc Dc H Ni, tr. 57-79.

7. Trn Vn n, Phng Thanh Hng (2011), "Sng lc cc dc liu c

tc dng iu tr i tho ng Vit Nam v nghin cu thnh phn

ha hc, c tnh, h ng huyt ca mt s dc liu in hnh",

ti khoa hc v pht trin cng ngh cp B.

8. L Thanh Phc, Bnh Nguyn Anh Ho (2011), "Gp phn kho st

thnh phn ho hc ca r cau (Areca catechu L.)", Trng i hc

Cn Th.

9. B Y T (1996), Quy ch nh gi tnh an ton v hiu lc thuc c

truyn,Ph lc 3: Hng dn v kho st c tnh ca thuc c truyn,

Quyt nh 371/BYT-Q.

10. V Th Thanh Thy (2013), Nghin cu s a dng cc loi trong chi

Gymnema R.Br. s dng ch th phn t ITS, Kho lun tt nghip dc

s i hc. Th vin trng i hc Dc H Ni.

11. Nguyn Ngc T (2013), Nghin cu du vn tay ho hc ca cc loi

trong chi Gymnema R.Br. Vit Nam, Kho lun tt nghip dc s i

hc. Th vin Trng i hc Dc H Ni.

12. Phm H Thanh Tng (2012), Nghin cu tnh a dng thc vt v

thnh phn ho hc ca cc loi trong chi Gymnema R.Br. Vit nam,

Lun vn thc s. Th vin trng i hc Dc H Ni.

TING ANH

13. Ali H, Houghton PJ, Soumyanath A (2006), Alpha-Amylase

inhibitory activity of some Malaysian plants used to treat diabetes with

particular reference to Phyllanthus amarus,J. Ethnopharmacol,

107:449455.

14. A.K. Jamal W.A. Yaacob, Laily B.Din (2008), "A Chemical study on

Phyllanthus reticulatus", pp. School of Chemical Sciences and Food

Technology, Faculty of Science and Technology, Universiti

Kebangsaan Malaysia, Malaysia.

15. Der Vorgelegt (1964), "Isolation and Characterization of

Phytoconstituents from Myanmar Medicinal Plants", Mathematisch-

Naturwissenschaftlich-Technischen Fakultt der Martin-Luther-

Universitt Halle-Wittenberg.

16. Kitajima Junichi, Kaoru Kimizuka, and Yasuko Tanaka (1999), "New

dammarane-type acetylated triterpenoids and their related compounds

of Ficus pumila fruit".

17. Liu Willow JH, ed., (2011), "Traditional Herbal Medicine Research

Methods: Identification, Analysis, Bioassay, and Pharmaceutical and

Clinical Studies", John Wiley & Sons, Inc., New Jersey, pp.

18. Liu J. P., Lu D., Li P. Y. (2012), "A novel hexanordammarane

glycoside from the leaves and stems of Panax quinquefolium L", Nat

Prod Res, 26(8), 744-748.

19. Lucetti Daniel L (2010), "Anti-inflammatory effects and possible

mechanism of action of lupeol acetate isolated from Himtanthus

drasticus (Mart.) Plumel", pp.

20. Meve Ulrich; Alejandro, Grecebio Jonathan (2013), "Taxonomy of the

Asian Gymnema latifolium (Apocynaceae, Asclepiadoideae,

Marsdenieae), including lectotypification of the synonymous G.

khandalense", Botanic Garden and Botanical Museum Berlin-Dahlem,

43, 81-86.

21. Na M, Kim BY, Osada H, Ahn JS (2009), Inhibition of protein

tyrosine phosphatase 1B by lupeol and lupenone isolated from Sorbus

commixta, J. Enzym Inhib. Med. Chme, 24:10561059.

22. Narvaez-Mastache JM, Garduo-Ramrez ML, Alvarez L, Delgado G

(2006), Antihyperglycemic activity and chemical constituents of

Eysenhardtia platycarpa, J. Nat. Prod, 69:16871691.

23. OECD/OCDE 407, (2008), OECD guidelines for the testing of

chemicals: Repeated dose 28 Day Oral Toxicity Study in Rodents.

24. Ogawa Y., Sekita K., et al. (2004), "Gymnema sylvestre leaf extract: a

52-week dietary toxicity study in Wistar rats", Shokuhin Eiseigaku

Zasshi, 45(1),8-18.

25. Ortiz-Andrade RR, et al. (2007), Alpha-Glucosidase inhibitory

activity of the methanolic extract from Tournefortia hartwegiana, an

anti-hyperglycemic agent, J. Ethnopharmacol., 109:4853.

26. Siddique H. R., Saleem M. (2011), "Beneficial health effects of lupeol

triterpene: a review of preclinical studies", Life Sci, 88(7-8), 285-93.

27. Takhtajan (2009), Flowering Plants, Springer Verlag, 521-529.

28. The-Plant-List (2010), "Version 1. Published on the Internet;

http://www.theplantlist.org/, pp. Retrieved, 1st January.

29. Ulbricht C., Abrams T. R., et al. (2011), "An evidence-based

systematic review of Gymnema (Gymnema sylvestre R. Br.) by the

Natural Standard Research Collaboration", J Diet Suppl, 8(3), pp. 311-

330.

30. Wang X., Wang W. (2013), "Cytotoxic and radical scavenging nor-

dammarane triterpenoids from Viburnum mongolicum", Molecules,

18(2), pp. 1405-17.

31. Yang F., Shi H., Zhang X., Yu L. L. (2013), "Two novel anti-

inflammatory 21-nordammarane saponins from tetraploid Jiaogulan

(Gynostemma pentaphyllum)", J Agric Food Chem, 61(51), 12646-

12652.

32. Yeh G. Y., Eisenberg D. M., Kaptchuk T. J., Phillips R. S. (2003),

"Systematic review of herbs and dietary supplements for glycemic

control in diabetes", Diabetes Care, 26(4), 1277-1294.

PH LC

Ph lc 1. Gim nh tn khoa hc ca mu nghin cu.

Ph lc 2. Ph ca cc cht phn lp.

GLHE9:

3-acetoxy22,23,24,25,26,27hexanordammaran20on

GLHE7: Lupeol acetat

Ph lc 2.1. Ph 1H-NMR , 13C-NMR, DEPT ca GLHE9

Ph 1H-NMR ca GLHE9

GLHE9CDCl31H

7 6 5 4 3 2 1 0 ppm

1.333

1.431

1.436

1.457

1.480

1.487

1.510

1.531

1.542

1.564

1.571

1.589

1.601

1.609

1.633

1.640

1.654

1.681

1.690

1.708

1.716

1.738

1.801

1.887

1.894

1.910

1.916

1.931

1.940

1.948

1.956

2.043

2.129

2.558

2.571

2.580

2.592

2.602

2.614

4.460

4.472

4.480

4.493

7.278

13.158

3.072

1.119

5.324

1.084

2.959

4.017

2.326

1.237

2.077

3.015

3.038

1.013

1.000

PC 1.00GB 0LB 0.30 HzSSB 0WDW EMSF 500.1300045 MHzSI 32768F2 Processing parameters

SFO1 500.1340010 MHzPL1 1.00 dBP1 10.30 usecNUC1 1H======== CHANNEL f1 ========

MCWRK 0.01500000 secMCREST 0.00000000 secD1 1.00000000 secTE 300.0 KDE 6.00 usecDW 50.000 usecRG 71.8AQ 3.2769001 secFIDRES 0.152588 HzSWH 10000.000 HzDS 0NS 16SOLVENT DMSOTD 65536PULPROG zg30PROBHD 5 mm MultinuclINSTRUM spectTime 16.27Date_ 20131220F2 Acquisition Parameters

PROCNO 1EXPNO 1NAME 110HOAI_GLHE9Current Data Parameters

Ph 13C-NMR ca GLHE9

GLHE9CDCl3C13CPD

220 200 180 160 140 120 100 80 60 40 20 ppm

16.260

16.457

18.099

21.206

21.267

23.656

25.540

25.934

27.936

30.006

31.516

35.438

37.081

37.865

38.757

40.475

45.092

50.019

50.589

54.219

55.928

76.762

77.017

77.271

80.829

170.969

212.329

PC 1.00GB 0LB 1.00 HzSSB 0WDW EMSF 125.7577921 MHzSI 32768F2 Processing parameters

SFO2 500.1320005 MHzPL13 22.00 dBPL12 16.81 dBPL2 1.00 dBPCPD2 80.00 usecNUC2 1HCPDPRG2 waltz16======== CHANNEL f2 ========

SFO1 125.7719472 MHzPL1 0.00 dBP1 6.10 usecNUC1 13C======== CHANNEL f1 ========

MCWRK 0.01500000 secMCREST 0.00000000 secDELTA 1.89999998 secd11 0.03000000 secD1 2.00000000 secTE 300.0 KDE 10.00 usecDW 15.900 usecRG 32768AQ 1.0420883 secFIDRES 0.479836 HzSWH 31446.541 HzDS 2NS 160SOLVENT CDCl3TD 65536PULPROG zgpg30PROBHD 5 mm MultinuclINSTRUM spectTime 16.35Date_ 20131220F2 Acquisition Parameters

PROCNO 1EXPNO 2NAME 110HOAI_GLHE9Current Data Parameters

Ph DEPT ca GLHE9

GLHE9CDCl3C13CPD

2030405060708090100110120130140150160170180190200210 ppm

2030405060708090100110120130140150160170180190200210 ppm

2030405060708090100110120130140150160170180190200210 ppm

DEPT90

DEPT135

C13CPD

CH&CH3

CH2

&DEPT

Ph lc 2.2. Ph 1H-NMR , 13C-NMR, DEPT ca GLHE7

Ph 1H-NMR ca GLHE7

GLHE7CDCl31H

8 7 6 5 4 3 2 1 0 ppm

1.311

1.320

1.328

1.340

1.348

1.363

1.379

1.387

1.393

1.396

1.416

1.458

1.467

1.472

1.484

1.489

1.493

1.497

1.508

1.512

1.560

1.571

1.586

1.603

1.610

1.619

1.626

1.632

1.648

1.656

1.684

1.699

1.709

1.894

1.911

1.917

1.921

1.939

2.038

2.168

2.346

2.358

2.368

2.380

2.390

2.402

4.455

4.466

4.476

4.487

4.569

4.684

4.688

7.261

4.066

9.473

3.272

2.277

3.214

1.049

5.133

6.953

2.316

2.357

9.076

1.068

3.088

0.876

1.011

1.033

1.004

1.000

PC 1.00GB 0LB 0.30 HzSSB 0WDW EMSF 500.1300137 MHzSI 32768F2 Processing parameters

SFO1 500.1335009 MHzPL1 1.00 dBP1 10.30 usecNUC1 1H======== CHANNEL f1 ========

MCWRK 0.01500000 secMCREST 0.00000000 secD1 1.00000000 secTE 300.0 KDE 6.00 usecDW 50.000 usecRG 114AQ 3.2769001 secFIDRES 0.152588 HzSWH 10000.000 HzDS 0NS 16SOLVENT CDCl3TD 65536PULPROG zg30PROBHD 5 mm MultinuclINSTRUM spectTime 9.36Date_ 20131110F2 Acquisition Parameters

PROCNO 1EXPNO 1NAME 110HOAI_GLHE7Current Data Parameters

Ph 13C-NMR ca GLHE7

GLHE7CDCl3C13CPD

200 180 160 140 120 100 80 60 40 20 0 ppm

16.193

16.507

18.023

18.230

19.309

20.972

21.322

23.738

25.138

27.464

27.968

29.868

34.247

35.599

37.117

37.824

38.080

38.422

40.023

40.885

42.856

43.018

48.030

48.327

50.380

55.417

76.769

77.023

77.278

81.011

109.362

150.978

171.022

PC 1.00GB 0LB 1.00 HzSSB 0WDW EMSF 125.7577877 MHzSI 32768F2 Processing parameters

SFO2 500.1320005 MHzPL13 22.00 dBPL12 16.81 dBPL2 1.00 dBPCPD2 80.00 usecNUC2 1HCPDPRG2 waltz16======== CHANNEL f2 ========

SFO1 125.7716224 MHzPL1 0.00 dBP1 5.20 usecNUC1 13C======== CHANNEL f1 ========

MCWRK 0.01500000 secMCREST 0.00000000 secDELTA 1.89999998 secd11 0.03000000 secD1 2.00000000 secTE 300.0 KDE 6.00 usecDW 15.900 usecRG 32768AQ 1.0420883 secFIDRES 0.479836 HzSWH 31446.541 HzDS 2NS 512SOLVENT CDCl3TD 65536PULPROG zgpg30PROBHD 5 mm MultinuclINSTRUM spectTime 9.49Date_ 20131110F2 Acquisition Parameters

PROCNO 1EXPNO 2NAME 110HOAI_GLHE7Current Data Parameters

Ph DEPT ca GLHE7

GLHE7CDCl3C13CPD

2030405060708090100110120130140150160170180 ppm

2030405060708090100110120130140150160170180 ppm

2030405060708090100110120130140150160170180 ppm

DEPT90

DEPT135

C13CPD

CH&CH3

CH2

&DEPT

LI CM NMC LCDANH MC CC K HIU V CH VIT TTDANH MC CC BNGDANH MC CC HNHT VN 1Chng 2. I TNG V PHNG PHP NGHIN CU.122.1. NGUYN VT LIU V HA CHT, DNG C NGHIN CU122.1.1. Nguyn vt liu 122.1.2. ng vt th nghim122.1.3. Dung mi, ho cht122.1.4. My mc - thit b13

2.2. NI DUNG NGHIN CU132.2.1. Nghin cu phn lp mt s cht tinh khit132.2.2. Xc nh c tnh bn trng din13

2.3. PHNG PHP NGHIN CU132.3.1. Phng php nghin cu phn lp mt s hp cht132.3.2. Phng php nghin cu v c tnh bn trng din15

Chng 3. KT QU NGHIN CU183.4. PHN LP MT S HP CHT183.4.1. Qu trnh chit xut183.4.2. Qu trnh phn lp193.4.3. Xc nh cu trc cc cht phn lp c20

3.5. C TNH BN TRNG DIN 243.5.1. nh hng ca dch chit l Dy tha canh l to n s tng trng th trng ca chut cng trng243.5.2. nh hng ca dch chit l Dy tha canh l to n cc ch s huyt hc ca chut cng trng253.5.3. nh hng ca dch chit l Dy tha canh l to n cc ch s sinh ho ca chut cng trng263.5.4. Kt qu v m bnh hc27

3.6. BN LUN293.3.1. V phng php nghin cu 293.3.2. V phn lp cc cht 303.3.3. V c tnh bn trng din32

KT LUN V XUT34TI LIU THAM KHOPH LCDANH MC CC K HIU V CH VIT TTDANH MC CC BNGDANH MC CC HNHT VN Chng 1. TNG QUAN1.1. LOI GYMNEMA LATIFOLIUM WALL. EX WIGHT1.1.1. V thc vt1.1.1.1. V tr phn loi1.1.1.2. c im thc vt ca loi

1.1.2. V a dng di truyn1.1.3. V thnh phn ho hc1.1.4. V tc dng sinh hc1.1.5. V c tnh

1.2. TNG QUAN V PHNG PHP NGHIN CU 1.2.1. V phn lp mt s hp cht1.2.1.1. Chit xut1.2.1.2. Phn lp v xc nh cu trc ho hc

1.2.2. V nghin cu c tnh bn trng din

Chng 2. I TNG V PHNG PHP NGHIN CU2.1. NGUYN VT LIU V HO CHT, DNG C NGHIN CU2.1.1. Nguyn vt liu 2.1.2. ng vt th nghim2.1.3. Dung mi, ho cht. 2.1.4. My mc - thit b

2.2. NI DUNG NGHIN CU 2.2.1. Nghin cu phn lp mt s cht tinh khit2.2.2. Xc nh c tnh bn trng din

2.3. PHNG PHP NGHIN CU2.3.1. Phng php nghin cu phn lp mt s hp cht2.3.1.1. Phng php chit xut* To dch chit ton phn* Chit xut cc phn on* Chit pha rn

2.3.1.2. Phng php phn lpSc k ct hp ph: Sc k ct c tin hnh trn ct silicagel pha thng (0,040 - 0,063 mm, Merck), silicagel pha o YMC (30 - 50 (m, FuJisilisa Chemical Ltd.).

2.3.2. Phng php nghin cu v c tnh bn trng din2.3.2.1. Chun b cao lng2.3.2.2. Th c tnh bn trng din. Hnh 2.1. Quy trnh th c tnh bn trng din ca dch chit Dy tha canh l to (Gymnema latifolium Wall. ex Wight)

Chng 3. KT QU NGHIN CU3.1. PHN LP MT S HP CHT3.1.1. Qu trnh chit xut.3.1.2. Qu trnh phn lp3.1.3. Xc nh cu trc cc cht phn lp c3.1.3.1. Hp cht 1 (GLHE9)

3.2. C TNH BN TRNG DIN 3.2.1. nh hng ca dch chit l Dy tha canh l to n s tng trng th trng ca chut cng trngTrong qu trnh nghin cu, chut cc l u n ung, hot ng bnh thng, mt sng, lng mt, phn kh, nc tiu bnh thng. Trong sut 28 ngy nghin cu, cn nng chut cng tng u cc l (Bng 3.3). Khng c s khc bit v khi lng chut gia l th v l chng ti tt c cc thi im nghin cu (p>0,05). Bng 3.3. nh hng ca dch chit l Dy tha canh l to n khi lng c th ca chut cng trng

3.2.2. nh hng ca dch chit l Dy tha canh l to n cc ch s huyt hc ca chut cng trng3.2.3. nh hng ca dch chit l Dy tha canh l to n cc ch s sinh ho ca chut cng trngBng 3.5. nh hng ca dch chit l Dy tha canh l to n cc ch s sinh ha ca chut cng trng

3.2.4. Kt qu v m bnh hc.

3.3. BN LUN3.3.1. V phng php nghin cu 3.3.2. V phn lp cc cht 3.3.3. V c tnh bn trng din

KT LUN V XUT