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P

NGHPHP

S

TR

HIN CP XC CH

V

KHA L

NG

L

CU C NT PH TH

LUN T

H

B Y I HCWY

C TU

XYNH HHN L

HN C

TT NG

H NI

T C DCZX

N T

Y DNM LLP

CY G

GHIP D

- 2013

C H N

NG PHLNGO

DC S

I

HNNG MC T

NG MT

B Y T TRNG I HC DC H NI

WY ZX

LC TUN T

NGHIN CU XY DNG PHNG PHP XC NH HM LNG MT

S CHT PHN LP C T V THN CY GO

KHA LUN TT NGHIP DC S

Ngi hng dn: 1. PGS.TS. Thi Nguyn Hng Thu 2. DS. Phm L Minh Ni thc hin: 1. Trung tm kim nghim Dc phm v M phm H Ni

2. B mn Ha phn tch c cht i hc Dc H Ni

H NI - 2013

LI CM N

Sau mt thi gian thc hin ti Nghin cu xy dng phng php xc

nh hm lng mt s cht phn lp c t v thn cy Go, ngoi s lm vic

nghim tc, s c gng, n lc ht mnh ca bn thn, ti nhn c rt nhiu s

khch l t pha nh trng, thy c, gia nh v b bn.

Trc ht, ti xin c by t lng bit n su sc ti PGS.TS. Thi Nguyn

Hng Thu Trng B mn Ha phn tch c cht, ngi dnh nhiu thi gian

v tm huyt hng dn nghin cu, gip ti hon thnh kha lun tt nghip.

Ti xin chn thnh cm n PGS.TS. Nguyn Thi An B mn Dc Liu,

ngi rt nhit tnh hng dn ti trong qu trnh thc hin ti.

ng thi ti xin gi li cm n ti ThS. H Th Thanh Huyn v DS. Phm

L Minh - B mn Ha phn tch c cht cng ton th cc cn b B mn Ha

phn tch-c cht, cn b Trung tm Kim nghim Dc phm v M phm H Ni

to mi iu kin thun li ti hon thnh kha lun ny.

Ti xin chn thnh cm n qu thy c Trng i hc Dc H ni, c bit

l nhng thy c trc tip ging dy ti sut thi gian hc tp ti Trng.

Cui cng, ti xin t lng bit n gia nh, bn b ng vin ti v mi mt

v gip ti tn tnh trong qu trnh hc tp, nghin cu, l ng lc khng nh

ti c kt qu ngy hm nay.

Ti xin chn thnh cm n!

H ni, thng 05 nm 2013.

Lc Tun T

MC LC

DANH MC CC K HIU, CC CH VIT TT

DANH MC BNG BIU

DANH MC HNH V, TH

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

1.1.TNG QUAN V CY GO...........................................................................2

1.1.1. c im thc vt......................................................................................2

1.1.2. Tnh v v cng nng ca cy Go ............................................................2

1.1.3. B phn dng, thu hi v ch bin ............................................................3

1.2.TC DNG DC L V THN CY GO ..............................................3

Hot ng antiangiogenic ca Lupeol ...........................................................4 Tc dng h huyt p .....................................................................................4 Tc dng chng vim.....................................................................................4 Tc dng bo v gan ......................................................................................4 Tc dng c ch s hnh thnh cc t bo tnh mch trung tm rn ca con ngi 4

Tc dng khng khun, khng nm ...............................................................51.3.CNG DNG CA V THN CY GO ....................................................5

1.4.THNH PHN HA HC V THN CY GO.........................................5

1.4.1. Cng thc ho hc, tnh cht vt l, tc dng dc l ca mt s cht phn

lp t v thn cy Go..........................................................................................6

1.5.MY SC K LNG HIU NNG CAO (HPLC) ......................................12

1.5.1. Khi nim sc k lng hiu nng cao......................................................12

1.5.2. Cc thng s c trng ............................................................................13

1.5.3. H thng my HPLC ...............................................................................15

1.5.4. Cc phng php nh lng bng k thut HPLC ................................16

1.5.5. Tng quan v sc k lng khi ph (LC-MS).........................................17

CHNG II - I TNG V PHNG PHP NGHIN CU ........................21

2.1. I TNG NGHIN CU .........................................................................21

2.2.THIT B V HA CHT.............................................................................21

2.2.1. Ha cht....................................................................................................21

2.2.2. Dng c thit b ........................................................................................21

2.3.NI DUNG NGHIN CU ............................................................................22

2.4. PHNG PHP NGHIN CU ...................................................................22

2.4.1. Chun b mu th ....................................................................................22

2.4.2. Chun b cc cht i chiu.....................................................................23

2.4.3. Kho st v tm iu kin sc k.............................................................23

2.4.4. Thm nh phng php phn tch..........................................................24

2.4.5. Phng php x l s liu.......................................................................26

CHNG III - THC NGHIM V KT QU......................................................27

3.1. HM M V HIU SUT CHIT CN TON PHN .............................27

3.1.1. Hm m ca dc liu:.............................................................................27

3.1.2. Hiu sut chit ..........................................................................................27

3.2. KHO ST IU KIN SC K.................................................................27

3.3. XC NH TINH KHIT CA CC HP CHT NGHIN CU......30

3.4. NH TNH CC HP CHT TRN SC K .....................................33

3.5. THM NH PHNG PHP NH LNG CC CHT TRONG V THN CY GO BNG HPLC...........................................................................36

3.5.1. Kim tra s ph hp ca h thng sc k: ...............................................36

3.5.2. Tnh c hiu ............................................................................................36

3.5.3. lp li ..................................................................................................37

3.5.4. Khong nng tuyn tnh ......................................................................37

3.5.5. ng ....................................................................................................41

3.5.6. Gii hn pht hin LOD v gii hn nh lng LOQ ............................41

3.6. NG DNG NH LNG CC CHT TRONG V THN CY GO 42

3.7.BN LUN .....................................................................................................42

3.7.1. V phng php x l mu .....................................................................42

3.7.2. V kho st iu kin sc k HPLC ........................................................43

3.7.3. V kt qu nh lng cc cht trong v thn cy Go...........................43

3.7.4. V thm nh phng php nh lng...................................................43

KT LUN V XUT........................................................................................45

KT LUN.............................................................................................................45

XUT ...............................................................................................................46

TI LIU THAM KHO

PH LC

DANH MC CC K HIU, CC CH VIT TT

C: Nng (g/mL)

ESI: Ion ha bng phun in t (Electronspray ionization)

EtOH: Ethanol

HPLC: Sc k lng hiu nng cao

LC-MS: Sc k lng ghp khi ph (Liquid Chromatography Mass

Spectrometry)

LOD: Gii hn pht hin (Limit of Detection)

LOQ: Gii hn nh lng (Limit of Quatification)

MeCN: Acetonitril

MeOH: Methanol

MS: Khi ph (Mass Spectrometry)

RSD: lch chun tng i (Relative Standard Deviation)

S/N: Tn hiu/nhiu ng nn (Signal/Noise)

SD: lch chun (Standard Deviation)

STT: S th t

S: Din tch pic sc k (mAu.s)

TP: Thnh phn

tR: Thi gian lu

UV-VIS: T ngoi kh kin (Ultraviolet visible)

DANH MC BNG BIU

Bng 3.1. m bt dc liu ......................................................................27

Bng 3.2. Mt s thng s th hin s ph hp ca h HPLC la chn ........36

Bng 3.3. Kt qu kho st lp li vi cc cht phn tch ............................37

Bng 3.4. Kt qu kho st tuyn tnh vi epicatechin.....................................38

Bng 3.5. Kt qu kho st tuyn tnh vi catechin .........................................38

Bng 3.6. Kt qu kho st tuyn tnh vi daucosterol.....................................39

Bng 3.7. Kt qu kho st tuyn tnh vi Lupeol ..........................................39

Bng 3.8. Kt qu kho st tuyn tnh vi Stigmasterol ...................................40

Bng 3.9. Kt qu kho st tuyn tnh vi friedelin .........................................40

Bng 3.10. Tng hp kt qu kho st ng ca 6 cht ...............................41

Bng 3.11. Kt qu kho st LOD v LOQ (g/mL) .......................................41

Bng 3.12. Kt qu xc nh hm lng tng cht trong mu v thn cy Go

nghin cu ...................................................................................................42

Bng ph lc. Kt qu kho st ng ca 6 cht trong v thn cy Go.

DANH MC HNH V, TH

Hnh 1.1. H thng my HPLC ............................................................................ 15

Hnh 1.2. S khi ca my khi ph ............................................................... 18

Hnh 1.3. S b t cc chp ba. ...................................................................... 19

Hnh 3.3. Sc k ca epicatechin phn lp c t v thn cy Go. ............ 31

Hnh 3.4. Sc k ca catechin phn lp c t v thn cy Go. ................. 31

Hnh 3.5. Sc k ca daucosterol phn lp c t v thn cy Go. ............ 31

Hnh 3.6. Sc k ca lupeol phn lp c t v thn cy Go. .................... 32

Hnh 3.7. Sc k ca stigmasterol phn lp c t v thn cy Go............ 32

Hnh 3.8. Sc k ca friedelin phn lp c t v thn cy Go. ................. 32

Hnh 3.9. Ph khi lng ng vi cc pic ca epicatechin trn sc k ca mu

th v hn hp cht i chiu .............................................................................. 33

Hnh 3.10. Ph khi lng ng vi cc pic ca catechin trn sc k ca mu

th v hn hp cht i chiu. ............................................................................. 34

Hnh 3.11. Ph khi lng ng vi cc pic ca daucosterol trn sc k ca

mu th v hn hp cht i chiu....................................................................... 34

Hnh 3.12. Ph khi lng ng vi cc pic ca lupeol trn sc k ca mu th

v hn hp cht i chiu..................................................................................... 34

Hnh 3.13. Ph khi lng ng vi cc pic ca stigmasterol trn sc k ca

mu th v hn hp cht i chiu....................................................................... 35

Hnh 3.14. Ph khi lng ng vi cc pic ca friedelin trn sc k ca mu

th v hn hp cht i chiu .............................................................................. 35

Hnh 3.15. Sc k ca mu trng (dung mi pha mu) ................................... 37

Hnh ph lc. Sc k ca hn hp mu th.

- 1 -

T VN

Cy Go (Bombax malabaricum DC.) l cy g cao n 15m, c hoa mu

ti, c bit n nh l mt cy thuc dn gian ti cc nc Pakistan, n ,

Trung Quc, Vit Nam v chu Phi. Theo kinh nghim dn gian, nhiu b phn ca

cy nh v thn, hoa v nha c s dng tr cc bnh nh vim lot d dy,

vim lot ngoi da, thp khp, b gy xng, kit l.

T v thn cy Go, nhm tc gi H Th Thanh Huyn v cng s

nghin cu vi mu thu hi ti H Ni, cc tc gi phn lp c mt s thnh

phn nh epicatechin [11], catechin, momor-cerebroside I [12], lupeol, stigmasterol

[13], friedelin v daucosterol.

Ngy nay, trn th gii xu hng tr v vi thin nhin, tm kim ngun

thuc mi v s dng thuc t tho dc ngy cng tng. Vit Nam, vi li th

v a hnh v kh hu to ra ngun ti nguyn cy c v cng phong ph cng

nh ngun dc liu di do cng vi tri thc s dng cy c lm thuc t lu i.

Tuy nhin nhiu loi cy c s dng rng ri theo kinh nghim dn gian m cha

c hoc c rt t nghin cu c gi tr khoa hc. Cn c cc nghin cu xc nh

hm lng cc hp cht tm thy trong dc liu nh gi ng tim nng ca

ngun dc liu v gp phn tiu chun ha nguyn liu trong qu trnh hin i

ha cc thuc c ngun gc dc liu. Do , ti Nghin cu xy dng

phng php xc nh hm lng mt s cht phn lp c t v thn cy

Go c thc hin nhm mc tiu:

1. Xy dng c mt quy trnh nh lng ng thi mt s hp cht

c phn lp t v thn cy Go bng phng php HPLC.

2. ng dng phng php xy dng c s b xc nh hm lng

epicatechin, catechin, daucosterol, lupeol, stigmasterol v friedelin c trong mt

mu v thn cy Go thu hi c.

- 2 -

CHNG I - TNG QUAN 1.1. TNG QUAN V CY GO

1.1.1. c im thc vt Tn khoa hc: Bombax malabaricum DC. h Go (Bombacaceae) [4], [18],

[19].

Tn ng ngha: Bombax ceiba L., Gossampinus malabarica (DC.) Merr.,

Salmalia malabarica (DC.) Schott et Endl, Bombax heptaphylla Cavl [4], [7], [8],

[9], [10], [14], [16], [18], [19].

Tn khc: Gn rng, Mc min th, My mn, My nghu (Ty) [4], [7], [8],

[9].

Cy g to, cao ti 15m hay hn. Thn cy thng, sn si, mu xm, c bnh

v to gc, l mc so le, kp chn vt, gm 5-7 l cht, hnh mc hoc hnh trng,

gc thun, u nhn, di 9-15cm, rng 4-5cm, hai mt nhn, mp nguyn, cung

chung di hn phin l, di t 20-25cm [2], [4], [7], [8], [9], [10], [18], [19].

Cm hoa mc u cnh thnh chm, 1-3 hoa mc cng mt cung. Hoa

mu , thnh thong mu cam, vng, ng knh 7,5-11cm, di 5-10cm. Hoa

nhiu, cung hoa ngn, nh, khe. Hoa to, u, lng tnh. Nh rt nhiu hp thnh

5 b hoc 6 b (khng thnh ng), ngn hn cnh hoa, b nm trong 2 cung cnh

khc nhau. Bu thng 5 , mt vi mang 5 u nhy, bu hnh nn, c lng mu

trng nht.

1.1.2. Tnh v v cng nng ca cy Go - Hoa Go c v ngt, tnh mt, c tc dng thanh nhit, li thp, tiu vim,

thu lim [3].

- V thn cy Go c v ng, tnh mt, c tc dng li tiu, tiu sng, gy

nn [17], khu phong, tr thp, tiu thng [3]. Theo ti liu n , nc sc v thn

c tc dng lm du vim, cm mu [3].

- R c v ng, tnh mt, c tc dng kch thch, b, cng c tc dng gy

nn v gim au [17], ngoi ra cn c tc dng thanh nhit, li thp, thu lim, ch

huyt, tn kt, ch thng. Indonesia, nc p r c tc dng h st [3].

- 3 -

- V t 15 16g, c th sc v ngm cha au rng [15]. V cn c dng

tr thp khp, au gip gy xng, dng cm mu trong cc chng bng huyt (phi

hp vi r non v ht cy ti), liu dng 15 30g [5], dng p cha tn nhang,

mn trng c, cc ri lon sc t da [10].

- L Go c dng rt tt cho i dt, pht ban, cha thiu mu, cha l

[10].

- Ht bng Go lm tng tit sa ph n sau khi sinh [17].

- Nha gm chch t thn cy Go c dng cha lu, kit l, cm mu,

rong kinh, kch dc [3].

- R Go p ly nc cha st, cha kit l, cha au d dy mn tnh [3],

cha au thng v, vim hch bch huyt dng lao v lm thuc li tiu. n ,

ngi ta dng r lm thuc kch dc cho trng hp bt lc, liu dng 30 50g

[17].

1.1.3. B phn dng, thu hi v ch bin Ton cy c s dng cho cc mc ch khc nhau:

- V thn: thu hi quanh nm, tt nht vo ma xun [2], [18], [19] nhng

cng c ti liu [6] cho rng tt nht vo ma h. V thn mang v co b v th

bn ngoi, ra sch, thi nh, phi kh sc ung, hoc dng ti gi nt p

ngoi [15], [4].

- R: thu hi vo ma xun hay ma thu, ra sch, thi nh, phi kh [15],

[4].

- Hoa: hi vo ma xun, p ly nc (dng ti) hoc phi kh, tn thnh

bt ung [15], [4], [18], [19].

- Gm, g, nha, ht, qu, bng: cng c thu hi, ch bin ph hp vi

mc ch s dng [15], [4], [18], [19].

1.2. TC DNG DC L V THN CY GO

Cy Go c bit n vi nhiu tc dng: chng vim, gim au, h huyt

p, h ng huyt, chng oxy ha, bo v gan, khng khun, khng nm, khng

Helicobacter pylori, h st, cm mu, tc dng c ch trn ng hnh thnh cc t

- 4 -

bo tnh mch rn ca con ngi, hot ng antiangiogenic, chng ung th v

HIV. Mt s tc dng dc l lin quan ti v thn cy Go nh:

Hot ng antiangiogenic ca Lupeol Nm 2003, Young-Jea You v cng s tin hnh sng lc mt s dc liu

thu hi ti Vit Nam c tc dng antiangiogenic cho thy dch chit methanol ca

v thn cy Go c tc ng antiangiogenic trn dng t bo thuc mng trong

ng dy rn (HUVEC). nng 30 v 50g/mL, lupeol c tc dng c ch trn

ng HUVEC vi t l tng ng l 40%-60% v trn 80%, trong khi n khng

nh hng ti s pht trin ca dng t bo ung th c nh SK-MEI-2, B16-F10

[31], [43].

Tc dng h huyt p Theo [43], lupeol v dch chit methanol ca v thn c tc dng h p.

Tc dng chng vim Chit xut v thn bng du ha thu c cn, cn c lc vi methanol thu

c cht kt tinh khng mu c xc nh l lupeol c tc dng chng vim [28],

[23], [21]. Cc phn tch thng k cho thy ba b phn gm v thn, li ca thn

cy v r c tc dng chng vim trn m hnh gy ph chn chut bng

carrageenan.

Tc dng bo v gan Theo [41], [23], cc b phn ca cy Go nh v thn, li thn, r c tc

dng gim nh tn thng gan gy ra bi CCl4. Tc dng gim mc hoi t t

bo, gim thoi ha cng c ghi nhn. Dch chit methanol ca mt s b phn

ca cy, cao lng 1g/mL cng c chng minh l c tc dng lm gim men gan,

gim s suy thoi v hoi t t bo gan thng qua th nghim gy c t bo gan

bng CCl4.

Tc dng c ch s hnh thnh cc t bo tnh mch trung tm rn ca con ngi

Do s hnh thnh mch qu nhiu cng l mt tin trnh bnh l thuc bnh

vim khp, thp khp nn Nguyn Hi Nam cng cc nh khoa hc Hn Quc

- 5 - tin hnh nghin cu tc dng c ch s hnh thnh cc t bo tnh mch trung tm

rn ca mt s cy thuc thu hi ti Vit Nam. Trong s 58 cy nghin cu th c 7

cy c tc dng c ch trong c cy Bombax malabaricum DC. [43].

Tc dng khng khun, khng nm Theo [28], dch chit nc, dch chit ethanol, dch chit ether ca bt v

thn c chng minh l c tc dng khng khun, vi cht i chiu ofloxacin.

Mu nghin cu c tc dng trn cc chng vi khun nh: Staphylococcus aureus,

Streptococcus pyogenes, Klebsiella pneumonia, Escherichia coli, Klebsiella

aerogenes, Neisseria gonorrhoae. Cc dch chit cng c tc dng khng nm, vi

cht i chiu fluconazole, chng nm th l Candida albicans.

1.3. CNG DNG CA V THN CY GO

V thn cy Go dng ti cha gy xng, sai khp, vim khp v mt s

bnh khc lin quan ti c xng khp. ng bo Ty Nguyn dng v Go v

v cy a qu to, phi kh, thi nh, sc ung cha bnh u ma. Nc sc c

ca v Go c ngm cha au rng [15], [4], [9], [18], [19]. V thn cn dng

cm mu trong chng bng huyt (phi hp vi r non v ht cy ti) [6], [7]. V

Go sao vng sc ung lm thuc an thn, li tiu vi liu 15-20g/ngy. V kh sc

ung 10-20g/ngy, dng ng thi gi p cha quai b. Nc sc v hoc hoa

dng cha a chy, kit l. V thn c s dng bn ngoi nh ming dn cho

vim nhim v pht ban ngoi da [15], [4], [9], [18].

1.4. THNH PHN HA HC V THN CY GO

H Th Thanh Huyn tin hnh nghin cu thnh phn ha hc ca v

thn cho thy:

V thn c cha: Glycosid Tim, saponin, flavonoid, coumarin, alkaloid,

tanin, acid hu c, ng kh, sterol. Khng c anthranoid, acid amin,

polysaccharid, cht bo, caroten [11], [12], [13], [34], [43]. Cng t v thn cy

Go, tc gi s dng sc k ct vi cht hp th l silicagel c c ht 40-63 m

phn lp c 2 cht: epicatechin v bis (2-ethylhexyl) adipate [11].

- 6 -

V thn cha cht nhy. Nm 2003, cc nh khoa hc Hn Quc phn lp

c mt cht t phn on dicloromethan l lupeol [33], [43]. Nm 1971, -

sitosterol cng c phn lp t v thn [32]. Ngoi ra, t v thn cy Go cn

phn lp c shamimicin [33], shamimin [28], 5-isopropyl-3-methyl-2,4,7

trimethoxy-8,1-naphtalen carbolacton v mt naphthoquinon l 8-formyl-7-

hydroxy-5-isopropyl-2-methoxy-3-methyl-1,4-naphthoquinon [32].

1.4.1. Cng thc ho hc, tnh cht vt l, tc dng dc l ca mt s cht phn lp t v thn cy Go

1.4.1.1. Lupeol

Ngun gc Nm 2013: H Th Thanh Huyn v cng s phn lp c lupeol [13] t

v thn cy Go vi hm lng khong 20mg trong 700g v thn kh.

Tnh cht vt l - Tn khoa hc: (1R, 3aR, 5aR, 5bR, 7aR, 9S, 11aR, 11bR, 13aR, 13bR)-3a,

5a, 5b, 8, 8, 11a-hexamethyl-1-prop-1-en-2-yl-1, 2, 3, 4, 5, 6, 7, 7a, 9, 10, 11, 11b,

12,13,13a, 13b-hexadecahydrocyclopenta [a]chrysene-9-ol

- Tng hp: Nm 2009 Surendra v Corey bo co mt con ng

tng hp Lupeol t (1E,5E)-8-[(2S)-3,3-dimethyloxiran-2-yl]-2,6-dimethylocta-1,5-

dienyl acetate [38].

- Cng thc phn t: C30H50O

- Khi lng phn t: 426,72 g/mol

- im nng chy: 212oC

- tan: tan tt trong MeOH, ether, benzene

- max: 350nm

- 7 -

Tc dng dc l - Nm 2003, Young-Jea You v cng s tin hnh sng lc mt s dc

liu thu hi ti Vit Nam c tc dng antiangiogenic cho thy dch chit methanol

ca v thn cy Go (c th l lupeol) c tc ng antiangiogenic trn dng t bo

thuc mng trong ng dy rn (HUVEC) [31], [43].

- Cng theo [41], [43], [23], lupeol cn c tc dng gim nh tn thng

gan gy ra bi CCl4. Tc dng gim mc hoi t t bo, gim thoi ha cng

c ghi nhn. Ngoi ra, lupeol cn c tc dng dn mch, h huyt p.

- Mt s nghin cu khc cng chng minh lupeol c tc dng chng vim

[42], [21], [20], [28], [23] v tc dng chng oxy ho, c th ng dng trong iu tr

ung th [20], [40].

1.4.1.2. Epicatechin

Ngun gc

Nm 2012, H Th Thanh Huyn v cng s thnh cng trong vic phn

lp Epicatechin t v thn cy Go [11], [13].

Tnh cht vt l - Tn khoa hc: (2R, 3R)-2-(3, 4-dihydroxyphenyl)-3, 4-dihydro-2H-

chromene-3, 5, 7-triol

- Cng thc phn t: C15H14O6


- im nng chy: 177oC

- tan: tan tt trong MeOH, EtOH

- max1: 220nm; max2: 277 nm

- 8 -

Tc dng dc l - Theo Katsuyuki Tanabe v cng s ti i hc Kyoto, Nht bn,

chng minh c rng epicatechin c tc dng hn ch tn thng thn do tc dng

bo v t bo ty th ca thn [39].

- Ngoi ra, trong nghin cu [34], Parmvir Bahia v cng s a ra rng

epicatechin c tc dng kch thch cc yu t p ng ERK thng qua AMP vng

iu chnh GluR2 trong t bo thn kinh v no, qua c tc dng ci thin dn

truyn thn kinh, ci thin tr nh.

- Ci thin cc chc nng v mch mu [35]. Thng 4 nm 2005, Hagen

Schroeter v cng s a ra rng, Epicatechin v cht chuyn ho ca n l

epicatechin-7-O-glucuronide u c tc dng ln ci thin mch mu ti ng mch

ch trn th. V cng chng minh c rng thc phm giu flavanol (Epicatechin)

pht huy li ch trn sc khe tim mch lm gim ri lon chc nng ni m, l mt

nguy c quan trng lin quan trong tin lng x va ng mch [35].

1.4.1.3. Catechin

Ngun gc

L ng phn quang hc ca Epicatechin. Nm 2012, H Th Thanh Huyn

cng phn lp c catechin t v thn cy Go [12].

Tnh cht vt l - Tn khoa hc: (2R, 3S)-2-(3, 4-dihydroxyphenyl)-3, 4-dihydro-1H-

chromene-3, 5, 7-triol



- im nng chy: 177oC

- tan: tan tt trong MeOH, EtOH

- 9 -

- max1: 209nm; max2: 279nm

Tc dng dc l - Nm 2009, Navindra P. Seeram v cng s th nghim tc dng ca

Catechin v Epicatechin trn c ch COX-1 v COX2 v cho thy kt qu c

ch tt ti nng 80M v hot ng c ch COX tt nht trong so snh vi cc

thuc c ch COX ibuprofen thng mi (ti 10M), naproxen (ti 10M ), Vioxx

(ti 1,67ppm) v Celebrex (ti 1,67ppm). nng 50mM, catechin c ch cc

t bo v, hiu qu chng li cc t bo rut kt, cc t bo phi. Cc t bo thn

kinh trung ng l nhy cm nht trong cc dng t bo th nghim. Nhn chung

catechin c tc dng c ch s gia tng ca cc dng t bo ung th [36].

- Gim s hp thu Aluminum trong tr ti d dy v rut non [37]. Ngoi

tc dng c ch COX v c ch s sinh trng ca t bo ung th, catechin cn

c chng minh c th s dng nh l mt cht lm gim lng Aluminum tch

lu trong c th do ung tr xanh, mt trong nhng nguy c gy c ang c

quan tm ti hin nay do tr l loi thc ung ang ngy cng ph bin trn th

gii. Nghin cu ch ra rng, catechin v cc polyphenol c tc dng gim s

hp thu Aluminum ti rut.

1.4.1.4. Daucosterol

Ngun gc:

Nm 2012, H Th Thanh Huyn v cng s cng phn lp c

Daucosterol t v thn cy Go [12].

Tnh cht vt l - Tn khoa hc: (3R, 4S, 5S, 6R)-2-[[(3S, 8S, 9S, 10R,13R, 14S)-17-[(2R,

5R)-5-ethyl-6-methylheptan-2-yl]-10, 13-dimethyl-2, 3, 4, 7, 8, 9, 11, 12, 14, 15,

- 10 - 16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-yl]oxy]-6-(hydroxymethyl)

oxane-3,4,5-triol



- Nhit nng chy: 284-286C

- tan: Tan tt trong MeOH

Tc dng dc l - Theo [26], tc dng dc l c bit ti ca daucosterol l iu ho

min dch c th, thng qua lm tng hot tnh ca t bo CD4 v Th1 ti chut qua

lm gim sinh trng ca t bo nm men Candida albicans. Trong nghin cu

ny, Lee JH v cng s ti i hc Dongduk Women a ra kt qu daucosterol

gip bo v t bo chut bi nm men Candida.

1.4.1.5. Stigmasterol

Ngun gc

Stigmasterol c tm thy trong v thn cy Go [13].

Ngoi ra, Stigmasterol cn c tm thy trong du u nnh vi nng

khong 0,54 g/mL [24] bng phng php HPLC.

Tnh cht vt l - Tn khoa hc: (3S, 8S, 9S, 10R, 13R, 14S, 17R)-17-[(E,2R,5S)-5-ethyl-6-

methylhept-3-en-2-yl]-10,13-dimethyl-2, 3, 4, 7, 8, 9, 11, 12, 14, 15, 16, 17-

dodecahydro-1H-cyclopenta[a]phenanthren 3-ol



- Nhit nng chy: 160 164oC

- 11 -

- tan: tan tt trong MeOH

- max: 257 nm

Tc dng dc l - Theo Richard E v cng s [25], phytosterols c tc dng lm gim

Lipoprotein t trng thp (LDL-C), l mt trong nhng ch s biu th mc

cholesterol mu. Mt k qu tng t cng c tm thy qua nghin cu [29] ca

Padmanabhan P Nair v cng s. Stigmasterol c tc dng gim s hp thu

Cholesterol vo mu, do lm gim nguy c bo ph v nguy c ung thu rut.

- Stigmasterol cn c dng lm nguyn liu tng hp progesterol [30].

Progesterol l mt hormon quan trng trong c th, progesterol c th dng mt

mnh trnh thai v iu tr nhiu loi bnh, bao gm xut huyt t cung bt

thng, v kinh, lc ni mc t cung, ung th v, thn, n mt ngon v st cn do

AIDS. Do , vic tng hp ra Progesterol mang tnh thc tin rt cao.

- Nguyn liu tng hp cortison [27]. Cortison cng l mt hormon c

tc dng gim au, dng trong cc bnh l min dch. Cng vi vic tng hp ra

Progesterol, tng hp cortison t Stigmasterol cng c rt nhiu ng dng trong

thc t.

1.4.1.6. Friedelin

Ngun gc

Nm 2012, H Th Thanh Huyn v cng s phn lp c t v thn cy

Go, ngoi ra friedelin cn c tm thy trong l cy Azima tetracantha L. [22].

- 12 -

Tnh cht vt l - Tn khoa hc: (4R, 4aS, 6aS, 6bR, 8aR, 12aR, 12bS, 14aS, 14bS) - 4, 4a, 6b,

8a, 11, 11, 12b, 14a-octamethylicosahydropicen-3(2H)-one


- Khi lng phn t: 426,72g/mol

- Nhit nng chy: 262-265oC

- tan: Tan tt trong MeOH, benzen, ether

- max: 287nm

Tc dng dc l Thng 8/2011, Paulrayer Antonisamy v cng s ch ra rng friedelin c

ch tnh thm acid acetic qua thnh mch ti chut. Friedelin c tc dng gim au

ng k (P

- 13 - 1.5.2. Cc thng s c trng [1]

H s phn b K H s phn b K ca cht phn tch c trng cho tc di chuyn ca cht

qua pha tnh v c xc nh bng:

K =

: Nng cht tan trong pha tnh.

: Nng cht tan trong pha ng

Nu = th qu trnh sc k s khng tch c hai cht A v B ra khi

nhau. cng nhiu th kh nng tch hai cht A v B trong qu trnh sc k

cng ln.

Thi gian lu (pht) L thi gian t lc tim mu vo h thng sc k n khi xut hin nh ca

pic (thi gian lu c trng cho mt cht). So snh thi gian lu ca mu th v

mu chun lm trong cng iu kin s nh tnh c cht .

K: H s dung lng

Trong mt php phn tch, nu nh qu th s tch km, cn qu ln

( >20 pht) th pic b dong v lp li km, thi gian phn tch di

H s dung lng K H s dung lng ca mt cht cho bit kh nng phn b ca cht trong

hai pha cng vi sc cha ca ct, tc l t s gia lng cht tan trong pha tnh v

lng cht tan trong pha ng trong thi im cn bng:

Trong v l lng cht tan A phn b trong pha tnh v

trong pha ng.

- 14 - S a l thuyt (N) v chiu cao a (H)

S a l thuyt biu th hiu nng ca ct trong mt iu kin sc k nht

nh. Mi a l thuyt trong ct sc k nh l mt lp pha tnh c chiu cao l H,

lp ny c tnh cht ng, tc l mt khu vc ca h phn tch m trong mt cn

bng nhit ng hc c thit lp gia nng trung bnh ca cht tan trong pha

tnh v trong pha ng.

S a l thuyt c tnh theo cng thc sau:

N = 16 hoc N = 5,54

Trong : : thi gian lu (pht).

W: chiu rng ca pic o y pic.

: chiu rng ca pic o na chiu cao ca nh.

Trong thc t N nm trong khong 2500 n 5500 l ph hp, ti thiu l 1000.

phn gii L i lng biu th tch ca cc cht ra khi nhau trn mt iu kin sc

k cho. phn gii ca 2 pic cnh nhau c tnh theo cng thc sau:

= hoc =

Trong :

, : thi gian lu ca 2 pic lin k ca 2 cht tan A v B (pht).

, : rng o cc y pic ca cht A v B (pht).

, : rng y pic na chiu cao pic ca cht A v B (pht).

Trong thc t nu cc pic cn i (Gauss) th phn gii ti thiu 2 pic

c ln bng nhau tch ra khi nhau l R=1,0.

H s bt i Cho bit mc cn i ca pic sc k, c tnh theo cng thc:

AF =

- 15 -

Trong :

: chiu rng pic c o 1/20 chiu cao pic.

a: khong cch t ng vung gc h t nh pic n mp ng cong pha

trc ca pic 1/20 chiu cao pic.

Trong php nh lng yu cu: 0,9 AF 2.

H s chn lc H s chn lc c trng cho tc di chuyn t i ca hai cht A v B

Theo qui c: < nn < 1.

Thng chn 1,05 2. Nu qu ln, thi gian phn tch s di.

1.5.3. H thng my HPLC [1]

Hnh 1.1. H thng my HPLC

My HPLC gm cc b phn sau:

- Bnh ng dung mi v h thng x l dung mi (Solvent Reservoir and

inlet Filters)

- Bm cao p, y pha ng qua ct sc k (Pump)

- B phn tim mu (Injector)

- 16 -

- Ct tch (Column)

- Detector

- My ghi tn hiu v x l s liu (Data System)

1.5.4. Cc phng php nh lng bng k thut HPLC [1] Tt c cc phng php nh lng bng sc k u da trn nguyn tc:

nng ca cht t l vi chiu cao hoc din tch pic ca n.

C 4 phng php nh lng thng c s dng trong sc k l:

- Phng php chun ngoi

- Phng php chun ni

- Phng php thm chun

- Phng php chun ha din tch

Trong khun kh ca kha lun ny ti xin trnh by c th v phng php

chun ngoi.

y l phng php nh lng c bn, trong c 2 mu chun v th u

c tin hnh sc k trong cng iu kin, so snh din tch (hoc chiu cao) pic

ca mu th vi din tch (hoc chiu cao) pic mu chun s tnh c nng ca

cc cht trong mu th.

C th s dng phng php chun ha 1 im hoc chun ha nhiu im.

Chun ha mt im Chn nng ca mu chun xp x vi nng ca mu th. Tnh nng

ca mu th theo cng thc:

Cx =

Cx: Nng mu th

CS: Nng mu chun

Sx: Din tch ca pic cht phn tch trong mu th

SS: Din tch ca pic cht phn tch trong mu chun

Chun ha nhiu im Cch tin hnh: Chun b 1 dy chun vi cc nng cht chun tng dn

ri tin hnh sc k. Cc p ng thu c l cc din tch hoc chiu cao pic mi

- 17 - im nng chun. V th biu din s tng quan gia din tch S (hoc chiu

cao H) ca pic vi nng ca cht chun. S dng on tuyn tnh ca ng

chun tnh ton nng ca cht cn xc nh. C th tnh theo 2 cch:

- p d liu din tch (hoc chiu cao) pic ca cht th vo ng chun s

suy ra c nng ca n.

- Xy dng phng trnh hi quy tuyn tnh m t quan h gia din tch

pic (hoc chiu cao) vi nng ca cht cn xc nh.

S = a + b.C

S: Din tch pic

a: Giao im ca ng chun vi trc tung

b: dc ca ng chun

C: Nng ca cht th.

Da vo phng trnh hi quy ny ta tnh c nng ca cht th.

Ch : ln ca din tch pic Sx (hoc chiu cao pic) mu th phi nm

trong on tuyn tnh ca ng chun [1].

1.5.5. Tng quan v sc k lng khi ph (LC-MS) [1] LC-MS l k thut phn tch da trn s kt hp sc k lng hiu nng cao

v phn tch khi ph. S kt hp ny to nn mt h thng c nhng ng dng

rng ln trong phn tch.

1.5.5.1. Nguyn tc

Khi ph (Mass Spectrometry MS) l k thut o trc tip t s khi lng

v in tch ion (m/z) c to thnh trong pha kh t phn t hoc nguyn t mu.

T s ny c biu th bng n v khi lng nguyn t hoc bng Dalton.

Cc ion c to thnh trong bung ion ha, c gia tc v tch ring nh

b phn tch khi trc khi n detector. Tn hiu tng ng vi cc ion s c

th hin bng mt s vch (pic) c cng khc nhau, tp hp thnh mt khi ph

- 18 - hoc ph khi, cung cp thng tin nh tnh, xc nh cu trc v nh lng

cc cht.

1.5.5.2. My khi ph

a) B np mu (Inlet)

a mu vo my. Nu mu dng lng hoc rn phi chuyn mu sang

dng hi bng cch thch hp. C th np mu trc tip hoc ni vi u ra ca mt

thit b phn tch khc nh sc k kh, sc k lng siu gii hn (SFC), in di mao

qun (CE).

Hnh 1.2. S khi ca my khi ph

b) B ngun ion (Ion source)

Ion ha cc phn t, nguyn t ca mu trng thi kh hoc hi, kh dung

mi a tip ion vo b phn tch khi, cch ly phn to ion p sut kh quyn

vi b phn nm trong chn khng su. V rt ra ngoi cc phn t trung ha, ion

khc du c th nh hng ti php o.

Cc k thut ion ha c pht trin v s dng l: Va chm electron, ion

ha ha hc, ion ha bng ngun ion phun sng kh solvat, ion ha ha hc p

sut kh quyn, ion ha bi ngun ion bng gii hp.

Trong ti ny, chng ti s dng k thut ion ha bng phun in t -

Electronspray ionization (ESI).

c) B phn tch khi (Mass Analyzer)

Tch cc ion theo t s m/z. Cc ion c gia tc v tch ring nh tc dng

ca t trng, in trng i n detector. C th phn b phn tch khi thnh 4

loi:

- B phn tch t (Magnetic Analyser )

- 19 -

- B phn tch t cc (Quadrupole)

- B phn tch thi gian bay (Time of Flight Analyser TOF)

- B phn tch cng hng ion cyclotron (Ion cyclotron resonance analyser

ICR)

ti ny chng ti s dng my khi ph c b phn tch khi l b phn

tch t cc chp ba.

Hnh 1.3. S b t cc chp ba.

B phn tch t cc chp 3 cn gi l Tandem Mass Analyser. K thut phn

tch khi ph kiu ny l khi ph 2 ln (MS/MS). K thut ny thng c dng

khng nh cu trc cc cht hu c v phn tch hn hp nhiu thnh phn cha

c tinh ch sch.

d) Detector

Detector c nhim v chuyn cc ion n thnh tn hiu in o bng h in

t ca my khi ph. Hin c 2 dng truyn thng:

- Nhn eclectron (electron myltiplier).

- Nhn quang (photomultiplier).

e) B x l d liu (Data System)

Tn hiu in t detector c khuych i trc khi chuyn thnh tn hiu

s phc v x l d liu theo yu cu khc nhau nh: ghi ph khi, so snh vi th

vin ph, nh lng

1.5.5.3. Mt s k thut LC-MS

- K thut phn tch ton thang (FULL SCAN)

Cho y thng tin v cc cht phn tch trong mu ion. Tuy nhin phng

php ny c nhy khng cao, nhiu ng nn c th ln.

- K thut phn tch chn lc ion (SIM, Selected Ion Monitoring)

K thut ny ch cho khi ph k nhn din mt ion v ghi sc theo thi

gian, k thut SIM nhy hn FULL SCAN t 10 ti 100 ln ty theo thit b.

- 20 -

- K thut MS/MS

Trong k thut MS/MS mt ion chn lc ch MS ln 1 c phn tch

bng cch s dng nng lng b gy, to ra mt hoc vi ion con c trng ch

phn tch MS ln 2. K thut MS/MS c s dng tng chn lc v tng

nhy do lm gim nhiu ng nn rt ng k.

Trong nghin cu ny, chng ti s dng k thut MS/MS nh tnh 6 hp

cht c trong v thn cy Go.

- 21 -

CHNG II - I TNG V PHNG PHP

NGHIN CU 2.1. I TNG NGHIN CU

Mu nghin cu l v thn ca cy Go (c lm kh t nhin) thu hi vo

ma h ti Hng Sn (M c, H Ni) v c TS. Trn Huy Thi (Vin

Sinh thi v Ti nguyn sinh hc) gim nh tn khoa hc l: Bombax malabaricum

DC., h Go (Bombacaceae).

2.2. THIT B V HA CHT

2.2.1. Ha cht

- Ha cht tinh khit phn tch: acid formic. - Dung mi loi dng cho HPLC: methanol, acetonitril. - Cc hp cht phn lp c t v thn cy Go l Lupeol, Friedelin,

Stigmasterol, Epicatechin, Catechin, Daucosterol c tinh ch v nhn dng cu

trc bng cc phng php ph MS v NMR.

2.2.2. Dng c thit b

- My sc k lng khi ph Shimadzu LC/MS 8030 c kt ni thm

detector SPD-20A (c kh nng phn tch UV-VIS trong vng 190-700nm) ca

Trung tm Kim nghim dc phm v m phm H Ni.

- Ct sc k Zorbax C18 (250mm x 4,6mm; 5m) kt hp bo v ct

Phenomenex C18 (ODS, Octadecyl; cartridge 1530mm ID).

- B lc dung mi, lc mu vi mng lc 0,45m

- B sinh hn hi lu v bnh cu

- Ni un cch thy

- Pipet t ng 1000l, 100l v 10l

- Cc dng c thy tinh khc: Bnh nh mc, pipet, ng ong, ng ly tm,

phu lc, mng lc

- My li tm Hettich ca c

- My o hm m Precisa XM 60, max 124g, d=0,001g

- 22 - 2.3. NI DUNG NGHIN CU

- nh gi tng tp c trong cc mu hp cht phn lp v tinh ch c

bng phng php chun ha din tch.

- Kho st cc iu kin sc k vi HPLC c th tch hon ton cc hp

cht trong hn hp 6 hp cht nghin cu vi nhau v vi cc hp cht khc trong

cao lng thn cy Go.

- nh tnh cc pic trn sc k da vo thi gian lu v kt hp vi MS

kt ni.

- Thm nh cc iu kin nh lng mt s hp cht trn vi cht i

chiu l cc cht phn lp c qua nhn dng cu trc v xc nh tng tp cht

bng phng php chun ha din tch.

- p dng s b xc nh hm lng 6 hp cht trn c trong mu thn

cy Go thu hi c.

2.4. PHNG PHP NGHIN CU

2.4.1. Chun b mu th

2.4.1.1. Xc nh m ca bt dc liu V thn sau khi sy kh, nghin thnh bt kh. Ly khong 2g bt mu

nghin cu xc nh m ca dc liu. Bt my o m, iu chnh nhit

110oC, rc bt dc liu ln a cn v tri u ln mt a, y a cn v i my

t ng hin kt qu ln mn hnh, c kt qu. Tin hnh 3 ln, ly kt qu trung

bnh.

2.4.1.2. Chit xut Qua th nghim vi nhiu bin php x l mu khc nhau, chng ti chn

phng php x l mu tng i ph hp nh sau: cn khong 500g dc liu v

cy ( xc nh m trc) c lm nh, ngm lnh 3 ln vi methanol trong

24 gi, 48 gi v 72 gi, thu dch chit, lc, ct thu hi dung mi di p sut gim.

Cn cn ton phn thu c v bo qun cn 4oC.

2.4.1.3. Tnh hiu sut chit Hm lng % ca cn ton phn so vi khi lng bt dc liu c tnh

- 23 - theo cng thc sau:

Trong : X: Hm lng (%)

M: Khi lng dc liu em chit (g)

x: m dc liu (%)

a: Khi lng cn ton phn thu c (g)

2.4.1.4. Pha dung dch th Dc liu sau khi chit, ct thu hi dung mi ti cn, cn chnh xc khong

0,5000g cn pha trong bnh 25mL, sau lc qua bng thy tinh, sau qua mng

lc 0,45m trc khi nh tnh v nh lng bng HPLC.

2.4.2. Chun b cc cht i chiu 2.4.2.1. Kim tra tinh khit ca cc hp cht nghin cu phn lp c

V cc hp cht nghin cu u tan tt trong methanol nn methanol c

chn lm dung mi pha dung dch i chiu.

Cc cht nghin cu c cn chnh xc, ha tan v nh mc thnh cc

dung dch trong methanol, lc qua mng lc v tim vo h thng sc k.

tinh khit ca cc hp cht nghin cu c nh gi qua tng tp c

trong chng sau khi c phn lp v tinh ch xc nh cu trc. Tng tp ca

cc hp cht c nh gi theo phng php chun ha din tch.

2.4.2.2. Pha cc dung dch i chiu gc Cn chnh xc mt lng tng cht nghin cu phn lp v tinh ch c,

cho vo bnh nh mc 25mL, thm va methanol, lc u cho tan hon ton.

Dung dch ny dng kim tra tng tp v c pha long thch hp xy dng

dy dung dch chun.

2.4.3. Kho st v tm iu kin sc k 2.4.3.1. Ct sc k

Qua tham kho cc ti liu nghin cu v phng php tch chit, nh

lng mt s thnh phn c trong cy Go [11], [12], [13], ti thy cc phng

- 24 - php u s dng sc k pha o. Hin nay, sc k phn b pha o l phng php

c s dng kh ph bin vi nhiu tnh u vit. Do , ti la chn sc k

phn b pha o trong nghin cu ny.

S dng ct hin c l Ct Zorbax C18 (250mm 4,6 mm; 5m) kt hp ct

bo v Phenomenex C18 (ODS, Octadecyl; cartridge 15x30mm ID).

2.4.3.2. Pha ng L mt trong nhng yu t quyt nh thi gian lu gi cc cht phn tch v

hiu qu ca s tch sc k.

Qua nghin cu tnh cht l ha ca cc cht v nghin cu mt s ti liu

tham kho, cc dung mi thng s dng l methanol, acetonitril, acid formic,

nc. Tin hnh kho st vi nhiu h dung mi khc nhau sao cho cc pic ca cc

hp cht nghin cu c tch ring r vi thi gian phn tch hp l.

2.4.4. Thm nh phng php phn tch 2.4.4.1. Kim tra s ph hp ca h thng

L chnh xc ca thit b, c xc nh bng cch o lp li nhiu ln

trn cng mt mu c x l xong.

Cch xc nh: Tin hnh tim lp li 6 ln dung dch chun chun b

trn, ghi li cc gi tr v thi gian lu, din tch pic. lp li ca h thng c

biu th bng lch chun tng i RSD.

2.4.4.2. Tnh c hiu Tnh c hiu l kh nng nh gi mt cch r rng cht cn phn tch khi

c mt cc thnh phn khc nh tp cht hoc cc cht cn tr khc.

Trong HPLC, tnh c hiu th hin: Trn sc k thu c t mu trng v

cc mu t to, pic ca cht cn phn tch tch hon ton vi cc pic tp.

Tin hnh:

Chun b cc mu sau:

- Mu trng

- Mu chun pha trong MeOH

- Mu th pha trong MeOH

- 25 -

So snh cc sc k thu c t vic phn tch cc mu trn.

Yu cu: ti thi gian lu ca cht chun khng xut hin pic l trn mu

trng, mu nn.

2.4.4.3. lp li ca phng php lp li ca mt phng php php phn tch l mc thng nht gia

cc kt qu ring bit khi quy trnh phn tch c p dng lp i lp li nhiu ln

trn cng mt mu ng nht.

lp li c biu th bng gi tr RSD

Tin hnh:

- Phn tch 1 mu 6 ln song song

- Xc nh kt qu nh lng theo ng chun, tin hnh trong cng iu

kin.

- Tnh lp li bng cch tnh lch chun tng i gia gi tr ca cc

ln nh lng.

2.4.4.4. Xc nh khong nng tuyn tnh tuyn tnh ca mt phng php phn tch: l s ph thuc tuyn tnh

gia i lng o c (y) v nng cht cn phn tch (x) trong khong xc

nh. N c biu th bng phng trnh hi quy y = ax + b v h s tng quan r.

Cch xc nh: kho st trn 1 dy cht chun c nng bin thin.

Xc nh s ph thuc tuyn tnh gia nng v din tch pic bng phng

trnh hi quy tuyn tnh.

Yu cu: H s tng quan r > 0,995.

2.4.4.5. ng L mc st gn cc gi tr tm thy trong phn tch so vi gi tr thc.

Cch xc nh: Tin hnh pha 1 dung dch chun, 6 dung dch th ring bit

theo quy trnh chun b dung dch chun v dung dch th. Thm vo mt mu th

mt lng cht chun sao cho tng nng ca chng vn nm trong khong tuyn

tnh kho st. ng s c tnh bng t l phn trm gia lng cht chun

tm c so vi lng cht chun thm vo.

- 26 -

Yu cu: tm li: 95% -105%

2.4.4.6. Gii hn pht hin LOD v gii hn nh lng LOQ Xc nh LOD v LOQ da vo t l p ng so vi nhiu ng nn (S/N),

t l S/N c tin hnh bng cch so snh p ng ca mu trng t tnh c

nng ti thiu ca cht phn tch c th pht hin c.

Tin hnh: Pha long dung dch chun t nng ban u n nng thp

nht c th pht hin c bng sc k sao cho vn nm trong khong tuyn tnh.

- LOD c xc nh bng nng ti thiu ca cht phn tch ti cho

cc pic c tn hiu bng 3 ln nhiu ng nn (S/N =3).

- LOQ l nng cht phn tch ti c tn hiu gp 10 ln nhiu ng

nn (S/N = 10).

2.4.5. Phng php x l s liu Cc kt qu sc k c x l bng cc phn mm Chemstation ca thit b

HPLC.

S dng cc phng php x l thng k trong phn tch vi cc i lng

c trng kt hp vi s h tr tnh ton ca Microsoft Office Excel.

- 27 -

CHNG III - THC NGHIM V KT QU 3.1. HM M V HIU SUT CHIT CN TON PHN

3.1.1. Hm m ca dc liu:

Ly 3 mu ngu nhin 3 v tr khc nhau ca mu dc liu em o hm

m bng my xc nh m Precisa XM 60. Kt qu thu c ti bng 3.1.

Bng 3.1. m bt dc liu

Th t Khi lng dc liu (g) m (%)

1 2,5463 7,33 2 2,4698 7,81 3 2,5129 8,02

Trung bnh 2,5096 7,72

3.1.2. Hiu sut chit

- Khi lng cn ton phn:

Cn 500,08g v thn cy Go (c m 7,72%) c lm nh, ngm lnh

3 ln vi methanol trong 24 gi, 48 gi v 72 gi. Thu hi dch chit, lc, gp dch

lc c ct thu hi dung mi di p sut gim thu c cn. Cn c c n

khi lng khng i, thu c 25,12g cn ton phn, em bo qun 4C.

- Hiu sut chit:

trong : M = 500,08(g)

x = 7,72%

a = 25,12(g)

Hiu sut X = 5,41% 3.2. KHO ST IU KIN SC K

Tin hnh kho st iu kin sc k vi ct hin c l Ct Zorbax C18

(250mm 4,6mm; 5m) kt hp ct bo v Phenomenex C18 (ODS, Octadecyl;

cartridge 15x30mm ID). Qu trnh kho st tin hnh nhit phng l 25C.

- 28 - S dng detector SPD-20A, chn bc sng pht hin 280nm dung ha

kh nng pht hin ca cc hp cht nghin cu.

Bt u t dng hn hp acetonitril v methanol c thm acid formic 0,1%

vi cc t l khc nhau ch ng dng u khng cho kh nng tch c 6 cht

nghin cu hoc thi gian phn tch qu di. Do vy chng ti phi s dng ch

gradient dung mi vi knh A l acid formic 0,1% trong acetonitril v knh B l

acid formic 0,1% trong methanol.

Sau khi th nghim vi mt s chng trnh dung mi khc nhau, tc

dng v th tch tim mu khc nhau, chng ti chn la c mt chng trnh

tng i ph hp tch cc cht nghin cu cng nh cc pic khc trong mu

nghin cu c chun b t v thn cy Go. Mt khc thi gian phn tch khng

qu ko di, l 30 pht cho mt ln tim mu. iu kin sc k chng ti xc nh

c vi iu kin ct sn c trn nh sau:

S dng ch gradient dung mi vi knh A l acid formic 0,1% trong

acetonitril v knh B l acid formic 0,1% trong methanol (B). T l knh A-B ban

u l (30:70), thay i tuyn tnh t n AB (40:60) pht th 6, ri thay i

tuyn tnh t n AB (50:50) pht th 23. Thay i tuyn tnh v li AB

(40:60) pht th 27 v cui cng pha ng c thay i tuyn tnh v t l AB

(30:70) ban u pht th 30.

Tc dng l 0,7 mL/pht v th tch tim mu l 10l.

Vi iu kin sc k nh trn, cc cht c tch hon ton, pic cn i, thi

gian lu hp l. Cc sc k thu c nh hnh 3.1 v hnh 3.2.

- 29 -

Hnh 3.1. Sc k ca hn hp 6 cht nghin cu.

Hnh 3.2. Sc k ca mu th chun b t v thn cy Go.

Tin hnh sc k vi tng hp cht nghin cu (hnh 3.3 n hnh 3.8) v so

snh vi sc k thu c ca hn hp 6 hp cht l epicatechin, catechin,

daucosterol, lupeol, stigmasterol v friedelin (hnh 3.1) xc nh c thi gian lu

ca cc cht ln lt l 4,375; 5,411; 7,878; 11,356; 17,935 v 21,348 pht.

- 30 -

Nh vy, qua qu trnh kho st, chng ti la chn c iu kin sc k

nh sau nh lng 6 cht: epicatechin, catechin, daucosterol, lupeol,

stigmasterol v friedelin:

Ct: Zorbax C18 (250mm 4,6mm; 5m) kt hp ct bo v

Phenomenex C18 (ODS, Octadecyl; cartridge 15x30mm ID), nhit 25.

Pha ng: S dng ch gradient dung mi vi knh A l acid formic

0,1% trong acetonitril v knh B l acid formic 0,1% trong methanol (B). T l

knh A-B ban u l (30:70), thay i tuyn tnh t n AB (40:60) pht th 6,

ri thay i tuyn tnh t n AB (50:50) pht th 23. Thay i tuyn tnh v

li AB (40:60) pht th 27 v cui cng pha ng c thay i tuyn tnh v t

l AB (30:70) ban u pht th 30.

Detector SPD-20A vi = 280nm.

Tc dng: 0,7 mL/pht.

Th tch tim: 10l.

3.3. XC NH TINH KHIT CA CC HP CHT NGHIN CU

Cc cht nghin cu c cn chnh xc v ha tan thnh cc dung dch

trong methanol v tim vo h thng sc k.

Thc hin chng trnh sc k nh mc 3.2, thu c sc k ca 6 hp

cht nh hnh 3.3 n hnh 3.8.

- 31 -

Hnh 3.3. Sc k ca epicatechin phn lp c t v thn cy Go.

Hnh 3.4. Sc k ca catechin phn lp c t v thn cy Go.

Hnh 3.5. Sc k ca daucosterol phn lp c t v thn cy Go.

- 32 -

Hnh 3.6. Sc k ca lupeol phn lp c t v thn cy Go.

Hnh 3.7. Sc k ca stigmasterol phn lp c t v thn cy Go.

Hnh 3.8. Sc k ca friedelin phn lp c t v thn cy Go.

- 33 - Cc sc k cc hnh 3.3 n hnh 3.8 cho thy c 6 hp cht u khng

xut hin pic tp cht (trong thi gian sc k l 30 pht) nn c th s dng cc hp

cht ny lm nguyn liu thit lp cht i chiu. Tuy nhin do lng cc hp

cht phn lp c cn t v cha thi gian cho php thit lp cht chun, do

vy chng ti tm dng chng nh cht i chiu kho st phng php nh

lng chng trong v thn cy Go.

3.4. NH TNH CC HP CHT TRN SC K

nh tnh bng so snh ph khi lng ca cc pic cng thi gian lu ca

mu th v hn hp cht i chiu.

Ph khi lng nhn dng cc pic c o ch : ESI+ vi tc kh

phun l 3 L/pht, tc kh lm kh l 15L/pht, nhit mao qun l 250oC, nhit

ca heat block l 400oC, vng qut m/z l 100-1000. Th ph mnh +4,5kV.

Ph khi lng ca cc pic tng ng thu c nh cc hnh 3.9-3.14.

Hnh 3.9. Ph khi lng ng vi cc pic ca epicatechin trn sc k ca mu

th v hn hp cht i chiu

- 34 -

. Hnh 3.10. Ph khi lng ng vi cc pic ca catechin trn sc k ca mu th

v hn hp cht i chiu.

Hnh 3.11. Ph khi lng ng vi cc pic ca daucosterol trn sc k ca mu

th v hn hp cht i chiu.

Hnh 3.12. Ph khi lng ng vi cc pic ca lupeol trn sc k ca mu th

v hn hp cht i chiu.

- 35 -

Hnh 3.13. Ph khi lng ng vi cc pic ca stigmasterol trn sc k ca mu

th v hn hp cht i chiu

Hnh 3.14. Ph khi lng ng vi cc pic ca friedelin trn sc k ca mu th

v hn hp cht i chiu

Kt qu hon ton ph hp gia ph khi lng ca cc pic tng ng vi

tng cht trn sc k hn hp 6 cht nghin cu v mu th l dch chit ton

phn chun b t v thn cy Go. Ngoi pic ca 6 hp cht nghin cu, trn sc k

ca mu th chun b t v thn cy Go cn xut hin thm 2 pic khc

khong 14,0 pht v 25,4 pht (hnh 3.2)

- 36 - 3.5. THM NH PHNG PHP NH LNG CC CHT TRONG V

THN CY GO BNG HPLC

3.5.1. Kim tra s ph hp ca h thng sc k:

Tin hnh tim lp li 6 ln dung dch chun chun b trn, ghi li cc gi

tr v thi gian lu. lp li ca h thng c biu th bng lch chun tng

i RSD ca thi gian lu.

Bng 3.2. Mt s thng s th hin s ph hp ca h HPLC la chn

Hp cht Epicatechin Catechin Daucosterol Lupeol Stigmasterol Friedelin

1 4,370 5,401 7,890 11,380 17,910 21,350 2 4,375 5,412 7,866 11,365 17,980 21,340 3 4,380 5,413 7,869 11,346 17,902 21,397 4 4,369 5,411 7,883 11,353 17,923 21,372 5 4,376 5,423 7,886 11,334 17,950 21,314 6 4,381 5,408 7,875 11,357 17,945 21,317

Trung bnh 4,375 5,411 7,878 11,356 17,935 21,348

Thi

gian

lu

(pht)

RSD (%) 0,11 0,13 0,12 0,14 0,16 0,15

S a l thuyt 22320 23230 24550 34660 35140 32340 H s bt i 1,11 1,05 1,10 1,07 1,04 1,12

Nhn xt: Cc thng s thc nghim th hin s ph hp ca h thng sc k

la chn phn tch cc hp cht nghin cu.

3.5.2. Tnh c hiu

Tnh c hiu c kim tra bng cch tim ln lt dung mi pha mu,

dung dch th v dung dch i chiu vo h thng sc k, kt qu cho thy dung

dch chun v dung dch th tng ng cho pic c thi gian lu tng ng (hnh 3.1

v hnh 3.2), cn dung mi pha mu khng cho pic cc thi gian lu tng ng

(hnh 3.15).

- 37 -

Hnh 3.15. Sc k ca mu trng (dung mi pha mu)

3.5.3. lp li

Tim lp li 6 ln vi mt dung dch nng kho st i vi mi cht

nghin cu vo h thng sc k kho st lp li.

Bng 3.3. Kt qu kho st lp li vi cc cht phn tch

Din tch pic (mAU.s) Hp cht

1 2 3 4 5 6 Tr.bnh

RSD (%)

Epicatechin 38872 38543 38590 38650 38596 38652 38650,5 0,27 Catechin 124346 124367 123454 124356 123243 124562 124054,7 0,41 Daucosterol 6184 6170 6155 6156 6243 6182 6181,67 0,48 Lupeol 60352 60367 60454 60356 60243 60862 60439 0,33 Stigmasterol 6351 6270 6325 6356 6343 6362 6334,5 0,49 Friedelin 21501 21470 21454 21356 21243 21262 21381 0,47

Nhn xt:

Kt qu kho st cho thy phng php HPLC chn c chnh xc cao

vi cc gi tr lch chun thu c tng i nh.

3.5.4. Khong nng tuyn tnh

Tim dung dch chun trong khong nng kho st vo h thng sc k

kho st tuyn tnh, kt qu c ghi trong bng 3.4 -3.9.

- 38 -

Bng 3.4. Kt qu kho st tuyn tnh vi epicatechin

Nng C(g/mL) 4,6 9,2 18,4 27,5 36,7 45,9

Din tc pic S (mAu.s) 7360 14718 29437 44154 58872 73590

Phng trnh hi quy vi epicatechin xc nh c l S = 1604,63.C -

34,73. Phng trnh ng chun tuyn tnh trong khong nng kho st 4,6-

45,9g/mL vi h s tng quan r l 1,0000. Bng 3.5. Kt qu kho st tuyn tnh vi catechin

Nng C(g/mL) 3,8 7,6 15,2 22,8 30,4 45,6

Din tc pic S (mAu.s) 16213 32420 64000 97201 129700 197023

Phng trnh hi quy vi catechin xc nh c l S = 4321,06.C-884,04.

Phng trnh ng chun tuyn tnh trong khong nng kho st 3,8-

45,6g/mL vi h s tng quan r l 0,9999.

- 39 -

Bng 3.6. Kt qu kho st tuyn tnh vi daucosterol

Nng C (g/mL) 1,7 5,1 13,6 20,4 27,2 34,0

Din tc pic S (mAu.s) 3573 10652 27420 42630 56841 71051

Phng trnh hi quy vi daucosterol xc nh c l S = 2093,72.C -

232,08. Phng trnh ng chun tuyn tnh trong khong nng kho st 1,7-

34,0g/mL vi h s tng quan r l 0,9999. Bng 3.7. Kt qu kho st tuyn tnh vi Lupeol

Nng C (g/mL) 4,5 9,0 17,9 26,9 35,8 53,8

Din tc pic S (mAu.s) 9546 19090 38180 57270 76001 118017

Phng trnh hi quy vi lupeol xc nh c l S = 2188,58.C 909,39.



- 40 -

Bng 3.8. Kt qu kho st tuyn tnh vi Stigmasterol

Nng C (g/mL) 2,4 7,2 9,6 14,4 19,2 24,0

Din tc pic S (mAu.s) 3504 10410 14000 21002 28020 34890

Phng trnh hi quy vi stigmasterol xc nh c l S = 1456,33.C- 3,36.


24,0g/mL vi h s tng quan r l 1,0000. Bng 3.9. Kt qu kho st tuyn tnh vi friedelin

Nng C (g/mL) 5,3 8,0 16,0 21,4 32,0 42,7

Din tc pic S (mAu.s) 7221 10801 21500 28700 43231 58500

Phng trnh hi quy vi friedelin xc nh c l S = 1367,76.C-260,66.



- 41 -

Nhn xt: Kt qu kho st cho thy c s tng quan tuyn tnh cht ch

gia nng cht phn tch v din tch pic p ng trong khong nng kho st

i vi cc hp cht nghin cu. Kt qu ny c s dng lm c s la chn

nng nh lng ca cc hp cht nghin cu tng ng.

3.5.5. ng Thm mt lng chnh xc dung dch chun vo dung dch th, sao cho tng

nng sau khi thm vn nm trong khong tuyn tnh ca phng php. Tin hnh

phn tch kt qu sc k kho st ng. Kt qu c tng hp trong bng

3.10, chi tit cho tng cht c th hin trong Ph lc.

Bng 3.10. Tng hp kt qu kho st ng ca 6 cht

T l thu hi (%) Hp cht Nng thm vo mu

(g/mL) Trung bnh min-max Epicatechin 7,34 99,20 97,44 - 99,84

Catechin 3,04 99,21 96,58 - 101,26 Lupeol 7,17 100,88 99,82 - 101,74

Friedelin 4,27 99,77 95,33 - 102,68 Stigmasterol 1,92 100,61 99,07 - 102,43 Daucosterol 1,36 101,42 99,97 - 102,19

Nhn xt: Kt qu kho st cho thy phng php phn tch la chn cho

t l thu hi cao, c ng tt.

3.5.6. Gii hn pht hin LOD v gii hn nh lng LOQ

Xc nh LOD da vo t l p ng so vi nhiu ng nn (S/N). Vic xc

nh t l S/N c tin hnh bng cch so snh p ng ca mu trng t tnh

c nng ti thiu ca cht phn tch c th pht hin c.

Bng 3.11. Kt qu kho st LOD v LOQ (g/mL)

Epicatechin Catechin Daucosterol Lupeol Stigmasterol Friedelin

LOD 0,5 0,5 0,34 0,61 0,32 0,8

LOQ 1,25 1,25 0,80 1,43 0,91 1,6

- 42 -

Nhn xt: Kt qu kho st cho thy phng php HPLC c nhy cao,

gii hn nh lng LOQ thp hn nng nh lng nhiu ln. Vic xc nh

LOQ ch yu gip cho vic phn tch tp cht.

3.6. NG DNG NH LNG CC CHT TRONG V THN CY GO

Tin hnh cn chnh xc khong 0,5000g cn ha tan v nh mc thnh

25mL, lc qua bng v qua mng lc. Tin hnh sc k song song mu th vi mu

hn hp cc cht i chiu. Tnh kt qu theo phng php chun ha 1 im.

Lng cn cn cho cc ln nh lng ln lt l: 0,5022g; 0,5032g v

0,5034g. Tnh ton lng tng cht c mu th qui v tng ng vi 0,5000g

cn. Tnh gi tr trung bnh ca 3 ln th. T lng dc liu v hm m ca n

em chit v tng lng cn thu c tnh ra hm lng tng cht c trong 100g

dc liu kh.

Kt qu nh lng thu c c trong 100g v thn cy Go c trnh by

bng sau:

Bng 3.12. Kt qu xc nh hm lng tng cht trong mu v thn cy Go nghin cu

Khi lng hp cht (mg) C trong cn em nh lng Tng ng trong 0,5g cn Hp cht Ln 1 Ln 2 Ln 3 Ln 1 Ln 2 Ln 3 Tr.bnh

Hm lng

(mg/100g dl)

Epicatechin 1,225 1,226 1,205 1,219 1,218 1,197 1,211 12,293Catechin 0,662 0,667 0,668 0,659 0,663 0,664 0,662 6,716Daucosterol 0,178 0,181 0,180 0,177 0,179 0,179 0,178 1,811Lupeol 0,936 0,935 0,939 0,932 0,929 0,933 0,931 9,451Stigmasterol 0,186 0,190 0,193 0,185 0,189 0,192 0,189 1,915Friedelin 0,752 0,751 0,746 0,749 0,746 0,741 0,746 7,566

Cn lm thm trn s lng mu ln c s liu chnh xc hn v hm

lng 6 cht trn c trong v thn cy Go.

3.7. BN LUN

3.7.1. V phng php x l mu nh lng cc cht trong cy Go t kt qu tt, trc ht cn phi chit

kit hot cht v lm sch dch chit. Qua tham kho cc ti liu, ti chn phng

php ngm lnh vi dung mi l methanol. y l phng php d thc hin trong

- 43 - iu kin phng th nghim, khng i hi trang thit b c bit m li cho hiu

sut cao.

3.7.2. V kho st iu kin sc k HPLC Da trn cc nghin cu trc y v cc ti liu cng b, kt hp vi cc

iu kin c sn ti tin hnh kho st cc iu kin sc k nh m t mc 3.2

v xy dng c chng trnh sc k trn my sc k lng hiu nng cao

HP1100. Vic nh gi thng qua xc nh tnh ph hp ca h thng sc k, kh

nng tch pic ra khi cht khc, hnh nh ca pic trn sc k v thi gian lu.

Kt qu l cc cht trong HPLC tch hon ton, pic cn i, thi gian lu khng

qu di. iu chng t, cc chng trnh sc k s dng trong ti l ph hp

nh lng cc thnh phn trong v thn cy Go.

3.7.3. V kt qu nh lng cc cht trong v thn cy Go 1. T mu v thn cy Go dng nghin cu chng ti xc nh c hm

lng ca 6 cht nghin cu c trong 100g dc liu nh sau: epicatechin l

12,293mg; catechin l 6,716mg; daucosterol l 1,811mg; lupeol l 9,451mg;

stigmasterol l 1,915mg v friedelin l 7,566mg.

2. Cn lm thm trn s lng mu ln c s liu chnh xc hn v

hm lng 6 cht trn c trong v thn cy Go.

3.7.4. V thm nh phng php nh lng Sau khi xy dng mt quy trnh phn tch, p dng quy trnh vo phn

tch trong thc t mt cch chnh xc, cho kt qu tin cy, cn thm nh li phng

php theo quy nh chung v phn tch nh lng.

- chn lc: Nh c phn mm x l th, chng ti ghi li cc pic

sc k v ch ra l pic cc cht thu c trn sc k l tinh khit. Chng t

phng php c chn lc rt cao.

- Khong nng tuyn tnh: Tin hnh kho st cc khong nng tuyn

tnh nh trong cc bng 3.4 n 3.9 cho thy cc cht u c ng hi quy trong

khong nng kho st c ng thng, phng trnh hi quy c h s tng quan

- 44 - >0,99 chng t s tng quan tuyn tnh nng ca cht v din tch pic trong

khong nng kho st.

- ng, lp li ca phng php: u cho kt qu ph hp vi yu

cu thc t cn phn tch.

- Gii hn nh lng: Vi cch chun b cc dung dch th trn th gii

hn nh lng (LOQ) v gii hn pht hin (LOD) c trnh by trong bng

3.11 mc 3.5.6.

Nh vy, chng trnh nh lng cc cht trong v thn cy Go bng

phng php HPLC m ti xy dng hon ton ng tin cy. ng thi phng

php c u im l n gin, nhanh chng, d thc hin, c th tit kim c thi

gian v chi ph kim nghim. Hin nay a s cc Trung tm kim nghim dc

phm, m phm n cc c s sn xut thuc u c trang b my HPLC. V vy,

kt qu nghin cu ca ti c th trin khai v ng dng vo thc t.

- 45 -

KT LUN V XUT KT LUN

Qua thi gian lm kha lun tt nghip, chng ti trin khai thc nghim

v thu c cc kt qu sau:

1. xy dng c mt phng php cho php nh lng ng thi 6

cht trong v thn cy Go bng sc k lng hiu nng cao vi cc iu kin sc k

c th nh sau:

- Ct pha o Zorbax C18 column (250; 4,6mm; 5m) kt hp bo v ct

Phenomenex C18 (ODS, Octadecyl) (5m, 3,0mm) ti 25oC.

- Tc dng: 0,7mL/pht.

- Th tch tim: 10l

- Detector: s dng detector SPD-20A, chn bc sng pht hin 280nm.

- Pha ng: S dng ch gradient dung mi vi knh A l acid formic

0,1% trong acetonitril v knh B l acid formic 0,1% trong methanol (B). T l

knh A-B ban u l (30:70), thay i tuyn tnh t n AB (40:60) pht th 6,

ri thay i tuyn tnh t n AB (50:50) pht th 23. Thay i tuyn tnh v

li AB (40:60) pht th 27 v cui cng pha ng c thay i tuyn tnh v t

l AB (30:70) ban u pht th 30.

2. Phng php xy dng c thm nh, kt qu cho thy cc ch tiu

u t yu cu, m bo phng php c th p dng vo nh lng cc cht c

trong v thn cy Go.

3. p dng phng php v xc nh ng thi c hm lng ca 6

hp cht trong 100g ca 01 mu v thn Go c th nh sau: epicatechin l

12,293mg; catechin l 6,716mg; daucosterol l 1,811mg; lupeol l 9,451mg;

stigmasterol l 1,915mg v friedelin l 7,566mg.

- 46 - XUT

- Tip tc phn lp v tinh ch thm cc hp cht c trong v thn cy Go

c th tin hnh thit lp cc cht chun i chiu. y l cng vic rt quan

trng c th cho kt qu xc nh hm lng cc cht trong v thn cy Go

c chnh xc v n nh hn.

- Do thi gian c hn, ti mi ch phn tch trn 01 mu v thn cy Go

nn cha c tnh i din cao. Do cn m rng nghin cu, nghin cu xc nh

hm lng hot cht trong v thn cy Go khc Vit c s liu chnh xc v

hm lng cc hp cht c trong l cy Go.

- Hon thin phng php, c th gp phn tiu chun ha dc liu cy

Go (Bombax Malabaricum DC.).

TI LIU THAM KHO TING VIT 1. Trn T An, Thi Nguyn Hng Thu (2006), Ho phn tch II, Trng i hc

Dc H Ni, 125-147, 173-223. 2. Huy Bch, Bi Xun Chng (1980), S tay cy thuc Vit nam, Nxb Y hc,

201. 3. Nguyn Huy Bch v cs (2004), Cy thuc v ng vt lm thuc Vit nam, tp I,

NXB Khoa hc v K Thut H Ni, 852. 4. B Y T (1978), Dc liu Vit Nam, Nxb Y hc, 5, 67-68. 5. V Vn Chi (1996), T in cy thuc Vit nam, Nxb Y hc, 508-509. 6. V Vn Chi (1997), T in cy thuc Vit nam, Nxb Y hc, 1437-1445. 7. V Vn Chi (2003), T in thc vt thng dng, Nxb Khoa hc v K Thut, tp

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PH LC

Kt qu kho st ng ca 6 hp cht c trong v thn cy Go Kt qu kho st ng vi Epicatechin

TT S mu

(mAU.s) S thm

(mAU.s) C thm

(g/mL) C c

(g/mL) C mi

(g/mL) % tm li

1 38872 50345 7,34 24,25 31,40 97,44 2 38543 50245 7,34 24,04 31,34 99,39 3 38590 50346 7,34 24,07 31,40 99,84 4 38650 50355 7,34 24,11 31,41 99,41 5 38596 50346 7,34 24,07 31,40 99,79 6 38652 50345 7,34 24,11 31,40 99,31 24,11 99,20

RSD (%)

0.27 0.08 0.00 0.28 0.07 0.82

Kt qu kho st ng vi Catechin

TT S mu

(mAU.s) S thm

(mAU.s) C thm

(g/mL) C c

(g/mL) C mi

(g/mL) % tm li

1 124346 136841 3,04 29,36 32,31 97,25 2 124367 137242 3,04 29,36 32,41 100,21 3 123454 136272 3,04 29,14 32,18 99,77 4 124356 136765 3,04 29,36 32,29 96,58 5 123243 136253 3,04 29,09 32,17 101,26 6 124562 137432 3,04 29,41 32,45 100,17 29,29 99,21

RSD (%)

0.41 0.32 0.00 0.42 0.32 1.71

Kt qu kho st ng vi Daucosterol

TT S mu

(mAU.s) S thm

(mAU.s) C thm

(g/mL) C c

(g/mL) C mi

(g/mL) % tm li

1 6184 9030 1,36 3,06 4,42 99,97 2 6170 9034 1,36 3,06 4,43 100,58 3 6155 9052 1,36 3,05 4,43 101,75 4 6156 9065 1,36 3,05 4,44 102,16 5 6243 9153 1,36 3,09 4,48 102,19 6 6182 9082 1,36 3,06 4,45 101,85 3,06 101,42

RSD (%)

0.48 0.46 0.00 0.44 0.44 0.83

Kt qu kho st ng vi Lupeol

TT S mu

(mAU.s) S thm

(mAU.s) C thm

(g/mL) C c

(g/mL) C mi

(g/mL) % tm li

1 60352 75841 7,17 28,36 35,65 101,60 2 60367 75842 7,17 28,37 35,65 101,51 3 60454 75672 7,17 28,41 35,57 99,82 4 60356 75665 7,17 28,37 35,56 100,42 5 60243 75753 7,17 28,31 35,61 101,74 6 60862 76132 7,17 28,60 35,78 100,16 28,40 100,88

RSD (%)

0.33 0.21 0.00 0.33 0.20 0.76

Kt qu kho st ng vi Stigmasterol

TT S mu

(mAU.s) S thm

(mAU.s) C thm

(g/mL) C c

(g/mL) C mi

(g/mL) % tm li

1 6351 9130 1,92 4,36 6,27 99,41 2 6270 9134 1,92 4,31 6,27 102,43 3 6325 9172 1,92 4,35 6,30 101,83 4 6356 9165 1,92 4,37 6,30 100,46 5 6343 9153 1,92 4,36 6,29 100,48 6 6362 9132 1,92 4,37 6,27 99,07 4,35 100,61

RSD (%)

0.49 0.18 0.00 0.47 0.22 1.19

Kt qu kho st ng vi Friedelin

TT S mu

(mAU.s) S thm

(mAU.s) C thm

(g/mL) C c

(g/mL) C mi

(g/mL) % tm li

1 21501 27500 4,27 15,93 20,31 102,68 2 21470 27242 4,27 15,90 20,12 98,79 3 21454 27372 4,27 15,89 20,22 101,29 4 21356 27165 4,27 15,82 20,07 99,42 5 21243 27153 4,27 15,74 20,06 101,14 6 21262 26832 4,27 15,75 19,82 95,33 15,84 99,77

RSD (%)

0.47 0.76 0.00 0.47 0.76 2.37

Sc k ca hn hp mu th

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