xác định adenosine trong thực phẩm chức năng có chứa đông trùng hạ thỏa bằng...

B Y T

TRNG I HC DC H NI

NGUYN TH VN ANH

XC NH ADENOSINE

TRONG THC PHM CHC NNG

C CHA NG TRNG H THO

BNG PHNG PHP HPLC

KHA LUN TT NGHIP DC S

H NI - 2014

B Y T

TRNG I HC DC H NI

NGUYN TH VN ANH

XC NH ADENOSINE

TRONG THC PHM CHC NNG

C CHA NG TRNG H THO

BNG PHNG PHP HPLC

KHA LUN TT NGHIP DC S

Ngi hng dn:

ThS. Bi nh Sn

Ni thc hin:

Vin KNVSATTP Quc gia

H NI - 2014

LI CM N

Vi tm lng bit n su sc, ti xin chn thnh cm n ThS. Bi nh Sn

giao ti v tn tnh hng dn, gip trong sut qu trnh thc hin kha lun.

Ti xin chn thnh cm n cn b, cng nhn vin Vin kim nghim V sinh

an ton thc phm Quc gia to mi iu kin thun li ti hon thnh ti.

Ti cng xin chn thnh cm n Ban gim hiu nh trng, cc phng ban,

cng cc ging vin, nhn vin B mn Ha phn tch - c cht v cc b mn

khc trong Trng i hc Dc H Ni gip ti trong sut qu trnh hc tp

ti trng.

Cui cng, ti xin chn thnh cm n gia nh, bn b quan tm ng vin

ti trong qu trnh hc tp v thc hin kha lun.

Ti xin chn thnh cm n!

H ni, ngy 09 thng 5 nm 2014

Sinh vin

Nguyn Th Vn Anh

MC LC

Trang

DANH MC CH VIT TT

DANH MC CC BNG

DANH MC HNH V V TH

T VN 1

Chng 1. TNG QUAN. 2

1.1. Gii thiu chung v ng trng h tho...................................................... 2

1.1.1. c im ca ng trng h tho.................................................. 2

1.1.2. Phn loi ng trng h tho......................................................... 2

1.1.3. Thnh phn ca ng trng h tho.. 3

1.1.4. Tc dng ca ng trng h tho.. 4

1. 2. Gii thiu v adenosine.. 6

1.2.1. Cu to ca adenosine 6

1.2.2. Chuyn ha v tc dng ca adenosine.......... 7

1.3. Phng php xc nh adenosine 8

1.3.1. Phng php sc k bn mng TLC.. 8

1.3.2. Phng php in di mao qun (CE)......................... 9

1.3.3. Phng php sc k lng... 9

1.4. Phng php sc k lng hiu nng cao (HPLC)........................ 11

1.4.1. Nguyn tc chung ca sc k lng 11

1.4.2. Phn tch nh tnh v nh lng bng HPLC.. 13

Chng 2. I TNG V PHNG PHP NGHIN CU................. 15

2.1. i tng ................................................................................................... 15

2.2. Ha cht v dng c .................................................................................. 15

2.2.1. Thit b........................................................................................... 15

2.2.2. Dng c......................................................................................... 15

2.2.3. Ha cht.......................................................................................... 14

2.3. Phng php nghin cu............................................................................. 16

2.3.1. Phng php ly mu.........16

2.3.2. Phng php phn tch......... 16

2.4. Thm nh phng php phn tch............. 17

Chng 3. THC NGHIM, KT QU V BN LUN.............................. 19

3.1. La chn phng php phn tch.................................................................... 19

3.2. Ti u iu kin xc nh adenosine trn h thng HPLC..............19

3.2.1. La chn ct tch sc k

3.2.2. Kho st thnh phn pha ng.......

3.2.3. Kho st chng trnh ra gii gradient............................................

3.2.4. iu kin ti u ca qu trnh sc k.................................................

19

21

23

26

3.3. Kho st quy trnh x l mu.......................................................................... 27

3.3.1. Kho st dung mi chit

3.3.2. Kho st dung mi loi bo.......

3.3.3. Kho st nhit chit.......

3.3.4. Kho st thi gian chit .....

3.3.5. Kho st s ln chit......

27

28

29

30

30

3.4. nh gi phng php phn tch............ 32

3.4.1. Kho st khong tuyn tnh v lp ng chun.......

3.4.2. Gii hn pht hin (LOD) v gii hn nh lng (LOQ) ca

phng php

3.4.3. nh gi c hiu ca phng php.............................................

3.4.4. nh gi lp li ca h thng........................................................

3.4.5. lp li v thu hi ca phng php.........................................

32

33

35

36

37

3.5. nh gi cht lng ca TPCN c cha THT..............................................40

3.6. Bn lun .......................................................................................................... 41

3.6.1. i tng nghin cu........................................................................ 41

3.6.2. Xy dng quy trnh phn tch............................................................ 41

3.6.3. Kho st quy trnh x l mu............................................................ 41

3.6.4. Thm nh phng php.................................................................... 42

3.6.5. Kt qu phn tch mu thc............................................................... 42

KT LUN V KIN NGH..............................................................................43

TI LIU THAM KHO

PH LC

DANH MC CH VIT TT

T vit tt Ting Anh hoc tn khoa hc Ting Vit

ACN Acetonitrile

AOAC Association of Official Analytical

Chemists

Hip hi cc nh Ha phn

tch

AMP Adenosine monophosphat

CE Capillary electrophoresis in di mao qun

CMC Carboxyl methyl cellulose

THT ng trng h tho

EtOH Ethanol

HPLC High performance liquid

chromatography

Sc k lng hiu nng cao

LOD Limit of detection Gii hn pht hin

LOQ Limit of quantification Gii hn nh lng

MeOH Methanol

MS Mass spectrometry Khi ph

PDA Photo diode array Dy diod quang

RSD Relative standard deviation lch chun tng i

SD Standard deviation lch chun

TB Trung bnh

TLC Thin layer chromatography Sc k lp mng

TPCN Thc phm chc nng

USFDA The united states food and drug

administration

Cc qun l thc phm v

dc phm M

USP The united states pharmacopeia Dc in M

UV Ultra violet T ngoi

Vis Visible Kh kin

DANH MC CC BNG

Bng 3.1. Kho st thnh phn pha ng.....

Trang

22

Bng 3.2. Kho st s ln chit mu................ 30

Bng 3.3 . Kho st khong tuyn tnh. 32

Bng 3.4. LOD v LOQ ca adenosine........................................................ 34

Bng 3.5. Kho st lp li ca h thng ................................................. 36

Bng 3.6. lp li v thu hi trn nn mu dng vin nang mm....... 38

Bng 3.7. lp li v thu hi trn nn mu dng vin nang cng ....... 38

Bng 3.8. lp li v thu hi trn nn mu dng lng......................... 39

DANH MC HNH V V TH

Trang

Hnh 1.1. Hnh nh ca Cordyceps sinensis.3

Hnh 1.2. Hnh nh ca Cordyceps militaris3

Hnh 1.3. Cu to ca mt s nucleoside trong ng trng h tho.. 4

Hnh 1.4. Cng thc cu to ca adenosine 7

Hnh 1.5. S cu to h thng HPLC..13

Hnh 1.6. Sc k trong phn tch sc k..13

Hnh 2.1. Quy trnh phn tch mu d kin. 17

Hnh 3.1. Ph hp th ca adenosine...........19

Hnh 3.2. Sc phn tch trn ct X-Brigde C18 20

Hnh 3.3. Sc phn tch trn ct Symmetry C18.20

Hnh 3.4. Sc phn tch trn ct IntertSustain C18 21

Hnh 3.5. Sc phn tch s dng pha ng H2O v ACN...22

Hnh 3.6. Sc phn tch s dng pha ng H2O v MeOH............22

Hnh 3.7. Sc phn tch theo chng trnh gradient 1.23



Hnh 3.10. Sc phn tch theo chng trnh gradient 4...25

Hnh 3.11. Sc phn tch theo chng trnh gradient 5.. 26

Hnh 3.12. Sc phn tch vi iu kin ti u.............27

Hnh 3.13. Kho st dung mi chit ....27

Hnh 3.14: Kho st nhit chit...29

Hnh 3.15: Kho st thi gian chit 30

Hnh 3.16. Quy trnh phn tch mu thc ...31

Hnh 3.17. Kho st khong tuyn tnh....32

Hnh 3.18. ng chun ca adenosine. 33

Hnh 3.19. Sc nn mu TPCN dng vin nang ti LOD............................... 34

Hnh 3.20. Sc nn mu TPCN dng lng ti LOD...................................... 34

Hnh 3.21. c hiu ca chun adenosine.......................................................35

Hnh 3.22. c hiu ca phng php trn nn mu dng nang cng............35

Hnh 3.23: c hiu ca phng php trn nn m dng nang mm..............36

Hnh 3.24: c hiu ca phng php trn nn mu dng lng.....................36

Hnh 3.25: nh gi cht lng mu TPCN c cha THT...............................40

-1-

T VN

Y hc c truyn Trung Quc t xa xa coi ng trng h tho l v thuc

c tc dng B ph ch can, b tinh in tu, ch huyt ho m, B ph ch

thn, h dng tng ph, T m trng dng, kh bnh kin thn, l loi thuc

c th cha c Bch h bch tn. Trn lm sng, cc nghin cu c truyn

cng nh cc thc nghim hin i u xc nh ng trng h tho hu nh khng

c tc dng ph i vi c th ngi v ng vt. Trong thnh phn ha hc ca

ng trng h tho, adenosine l mt trong nhng hot cht chnh c tc dng.

Adenosine c vai tr quan trng trong qu trnh chuyn ho nng lng ca c

th. N gip ci thin tun hon ngoi bin v tim mch, ci thin nng lc c bp,

gim sinh trng ca t bo xu, tng lng oxy trong mu. V vy, vic b sung

adenosine hm lng cao gip c th lun di do nng lng hot ng hiu

qu v nhanh chng xo i cc triu chng mt mi.

Tuy nhin, hin nay ti Vit nam cha c phng php chun xc nh hm

lng hot cht chnh trong ng trng h tho nn vic pht trin phng php

phn tch cc hot cht chnh ni chung v adenosine ni ring trong nguyn liu

hay cc sn phm c cha ng trng h tho l v cng cn thit. N l cng c

gip cho cc nh qun l kim sot tt cht lng v gip cho ngi tiu dng c

s dng cc sn phm m bo cht lng.

V vy, chng ti tin hnh nghin cu ti: "Xc nh adenosine trong

thc phm chc nng c cha ng trng h tho bng phng php HPLC"

vi cc mc tiu sau:

+ Xy dng phng php xc nh adenosine trong TPCN c cha THT

bng phng php sc k lng hiu nng cao HPLC

+ ng dng phng php phn tch sn phm TPCN c cha THT trn

th trng. T , nh gi cht lng sn phm TPCN c cha THT.

-2-

Chng 1. TNG QUAN

1.1. Gii thiu chung v ng trng h tho

1.1.1. c im ca ng trng h tho [10], [14], [21], [22], [23]

Cordyceps (ng trng h tho) l mt loi nm thuc ngnh Ascomycotina,

lp Asiomycetes, c coi l mt thn dc c gi tr cao trong y hc Trung Quc

trong nhiu th k. l mt dng cng sinh gia mt loi nm ti c tn khoa hc

l Cordyceps sinensis vi u trng ca mt loi cn trng thuc chi Hepialus.

Thng gp nht l loi Hepialus armoricanus, ngoi ra cn 40 loi khc thuc chi

Hepialus cng c th b Cordyceps sinensis k sinh.

Cordyceps xut pht t ting Latinh "Cord" = "chy", "ceps" = "u". Nm c

dng ging con su, vi ui l mt cnh nh, mc l. Khi sy kh c mi tanh nh

c, t ln c mi thm. Phn "l" hnh dng ging ngn tay, di khong 4 11 cm,

trng lng khong 0,06g do si nm mc dnh lin vo u su non m thnh. u

su non ging nh con tm, di chng 3 5 cm, ng knh khong 0,3 - 0,8 cm.

Bn ngoi c mu vng sm hoc nu vng vi khong 20 - 30 vn kha, vn kha

gn u nh hn, c 8 cp chn, nhng 4 i gia l r nht. Cht m nm hnh

que cong mc ra t mnh su non, di hn su non mt cht. Su non d b gy,

rut bn trong cng y, mu trng hi vng, cht m nm kh dai v bn trong

rut hi rng, c mu trng ng. Khi nm t n trng thnh nht, n tiu th

n 99% cht dinh dng t thn su v su bin thnh xc kh. Vo ma ng, su

b nhim bo t nm do n phi hoc ht phi bo t nm. Vo ma h, nm pht

trin thnh cy mc ra t u su vn ra khi mt t. ng trng h tho ch

c pht hin vo ma h mt s cao nguyn cao hn mt bin t 3500 n

5000m nh: Ty Tng, T Xuyn, Thanh Hi, Cam Tc, Vn Nam...

1.1.2. Phn loi ng trng h tho [14]

Chi nm Cordyceps c ti 680 loi khc nhau, ring Trung Quc tm thy

60 loi. Tuy nhin cho n nay ngi ta mi ch nghin cu nhiu nht c 2 loi

Cordyceps sinensis v Cordyceps militaris.

-3-

Hnh 1.1. Hnh nh ca Cordyceps sinensis

Hnh 1.2. Hnh nh ca Cordyceps militaris

1.1.3. Thnh phn ca ng trng h tho [7], [14], [18], [19], [21], [23]

Trong ng trng h tho cha nhiu hp cht c hot tnh sinh hc qu: cc

hp cht nucleoside, cc polysaccharide, protein v cc hp cht nitragenous, sterol,

vitamin, khong cht...

* Cc nucleoside

y l thnh phn chnh c gi tr c bit trong ng trng h tho, gm mt

s hp cht nh: deoxyuridine, 2-3-dideoxyadenosine, hydroxyethyladenosine,

cordycepin [3-deoxyadenosine], uridine, guanidine, deoxyguanidine, cordycepin

triphosphate. Trong , adenosine l mt nucleoside c hot tnh sinh hc chnh

nh gi cht lng ca ng trng h tho.

-4-

. Hnh 1.3. Cu to ca mt s nucleoside trong ng trng h tho

* Cc nhm hp cht khc

Nakamura v cng s [18], [19] nghin cu cho thy cc polysaccharide

trong THT c hiu qu trong vic iu tit ng trong mu, chng di cn, c

hiu qu chng ung th v tng cng min dch.

Cordyceps cha protein, peptide v 17 acid amin thit yu khc. Ngoi ra,

Cordyceps cn cha mt s dipeptide cyclic khng ph bin nh cyclo-[Gly-Pro],

cyclo-[Leu-Pro], , lng nh cc polyamin nh 1,3-diamino propane,. Cc hp

cht ny c chng minh l c tc dng khng sinh [14]

Mt s cc sterol c trong ng trng h tho nh ergosterol, ergosterol

peroxide,.... c tc dng chng ri lon tnh dc.

Cc vitamin cn thit cho c th: trong 100g ng trng h tho c 0,12mg

vitamin B12; 29,19mg vitamin A; 116,03mg vitamin C, ngoi ra cn c vitamin

B2, vitamin E, vitamin K.

Ngoi ra cn c hp cht hydrocarbon, alcohol v aldehyde,

1.1.4. Tc dng ca ng trng h tho [7], [14], [16], [23]

ng trng h tho c s dng kh ph bin trong khong 200 nm trc

y Trung Quc v cc nc Phng ng. n nm 1976, n c bit n

v s dng rng ri ti cc nc phng ty.

-5-

ng trng h tho c tc dng b phi, thn, cm mu, tan m, iu tr ho

mn tnh, iu tr ra m hi trm v m bo phc hi sc khe nhanh cho ngi b

bnh v tng cng sc khng.

Ci thin chc nng gan: chng x gan, vim gan mn tnh v cc bnh lin

quan n gan khc. Th nghim lm sng cho thy khi s dng TPCN c cha

THT trn 33 bnh nhn vim gan B v 8 bnh nhn x gan cho thy trn 71%

bnh nhn c ci thin tnh trng bnh l.

Ci thin chc nng thn: nghin cu Trung Quc cho thy 51% s bnh

nhn ci thin c bnh thn mn tnh khi s dng 1 thng cc sn phm t

Cordyceps.

Tng kh nng hp th O2, c kh nng lm gim bnh phi mn tnh

Ci thin chc nng tim: iu tr ri lon nhp tim v suy tim mn tnh

Bo v c th chng li cc gc t do

Gim kch thc ca khi u v chng li ung th: dng polysaccharide chit

xut t Cordyceps vi liu 1-10mg/kg trng lng mi ngy cho chut c khi u

cho thy c tc dng chng khi u sarcoma 180. Mizuno [16], nm 1999 nghin

cu dng -(1,3)-D-glucan, phn on CO-N, dn xut t THT C.ophioglossoides

chng li chui t bo sarcoma 180 vi liu 0,5mg/kg trn chut. Kt qu, khi u

b hn ch pht trin vi t l t n 98%. Trong mt nghin cu iu tr 50 bnh

nhn b ung th phi vi liu iu tr 6g/ngy cng vi ho tr, khi u gim xung

46%. Khi iu tr cc loi ung th khc nhau vi liu 6g/ngy trong vng hai thng

cho thy bnh c ci thin ng k, kim tra t bo bch cu thy tng cao cc

bnh nhn c khi u gim 50%.

iu tit kh nng min dch: khi cc bnh nhn b thiu kh nng min dch,

nh bnh nhn ung th, vim gan B v HIV c s dng ng trng h tho h

tr iu tr, kt qu cho thy s lng v tc ng ca bch cu trong mu tng.

Ngc li, vi bnh nhn tng min dch qu cao nh bnh bch cu lympho hoc

thp khp khi dng ng trng h tho cho thy s lng v tc ng ca bch cu

trong mu gim v s lng hng cu li tng.

-6-

Duy tr lng cholesterol tt: Theo mt s nghin cu C.sinensis c kh

nng gim lng cholesterol tng xung 10-21% v triglycerid xung 9-26% ng

thi lm tng hm lng cholesterol c t trng cao (HDL - High density

Lipoprotein) t 27 - 30%

H tr cc triu chng lo ha: Nghin cu lm sng trn mt s bnh nhn

ln tui b mt mi v cc triu chng lin quan n lo ha sau khi s dng cc sn

phm t ng trng h tho trong 30 ngy. Mt mi ca h gim 92%, cm gic

ca h v lnh gim 89% v hin tng chng mt gim 83%, mt s bnh nhn c

vn h hp c ci thin r rt.

Ci thin chc nng tnh dc

Mt s hp cht c hot tnh sinh hc c s dng nhiu trong lnh vc y hc

nh 2,3- dideoxyadenosine (c coi l cht antiretroviruses) dng cho iu tr ly

nhim HIV (cc bit dc: DidannosineTM hay VidexTM)

Cc nghin cu c truyn cng nh hin i u xc nh ng trng h

tho hu nh khng c tc dng ph i vi c th ngi v ng vt. Liu ung

THT an ton i vi chut th nghim l trn 45g/1kg th trng.

Vi nhng tc dng nh trn, ng trng h tho c coi l mt thn dc

c s dng rng ri trong y hc. Trong , adenosine l thnh phn hot cht

chnh nh gi cht lng ca ng trng h tho.

1. 2. Gii thiu v adenosine

1.2.1. Cu to ca adenosine [13], [20], [22]

Adenosine l mt nucleoside nhn purin. y l hp cht cha N c cu trc

phn t hai vng gm mt phn t adenine lin kt vi mt ng ribose nh lin

kt -N9-glycosidic.

Cng thc phn t C10H13N5O4, khi lng phn t 267,241g/mol

Tn thng mi: adenocard

Cng thc cu to ca adenosine:

-7-

Hnh 1.4. Cng thc cu to ca adenosine

Adenosine dng bt tinh th mu trng, tan nhiu trong nc. ha tan tng ln

khi nhit tng v pH gim.

1.2.2. Chuyn ha v tc dng ca adenosine [1], [7], [13], [20]

Trong c th, adenosine c to thnh do s thy phn AMP bi enzym 5'-

nucleotidase, mt phn b chuyn ha ngc tr li to thnh AMP bi enzym

adenosine kinase, mt phn chuyn ha thnh inosine nh enzym adenosine

deaminase, sau chuyn ha thnh hypoxanthine nh enzym nucleosidase v

thnh xanthin nh enzym xanthin oxydase, sn phm cui cng ca s chuyn ha

l to thnh acid uric.

Adenosine l mt nucleoside ni sinh nh hng nhiu qu trnh sinh l ca

c th, n c mt khp cc c quan trong c th v ng vai tr quan trng trong

qa trnh trao i cht ca c th nh chuyn ha nng lng cng nh truyn tn

hiu cho AMP vng.

* c tnh khng vim

Adenosine c chng minh l mt cht c kh nng khng vim ti th th

A2A. Nng adenosine ngoi bo t bo bnh thng l khong 300nM. Tuy

nhin, p ng vi tn thng t bo nng ny c nhanh chng nng ln

600 - 1200nM. V vy, chc nng chnh ca adenosine l ngn nga tn thng m

trong trng hp thiu oxy, thiu mu cc b, co git, tng cng tun hon mch

vnh, mch no v iu ha nhp tim

-8-

* Tc dng trn tim

Adenosine trc tip kim sot cc chc nng ca m tim, ng thi n c kh

nng bo v tim chng li mt s chuyn ha c hi bng cch gim s trao i cht

ti c tim v tng lu lng mu ng mch vnh.

Adenosine c coi l thuc iu tr hiu qu ri lon nhp nt nh tht, ri

lon nhp trn tht cp. Hi phc c nhp xoang ngi b ri lon nhp nhanh

trn tht kch pht.

* Tc dng trn phi

Adenosine c kh nng iu chnh chc nng ca nhiu t bo khc nhau

lin quan n vim ng h hp nh bch cu trung tnh, bch cu a acid, t bo

lympho v i thc bo.

* Tc dng trn h thng thn kinh trung ng

Adenosine c tc dng tt vi ri lon thn kinh nh: thiu no cc b, ng

kinh, cc bnh thoi ha thn kinh v au dy thn kinh. V vy, adenosine c

s dng nhiu trong y hc sn xut thuc dng iu tr ri lon nhp tim,

thuc chng lon nhp tim, bnh lin quan n h thng thn kinh...

* Tc dng trn tc

Adenosine c chng mnh c kh nng kch thch s pht trin ca tc

nhng ngi c tc tha

1.3. Phng php xc nh adenosine

1.3.1. Phng php sc k bn mng TLC [8], [15]

Ma Yan v cng s [15] tin hnh xc nh cc nucleoside trong Cordyceps

sinensis bng phng php TLC. Dch chit c phn tch trn bn mng silica

GF254 c cha 1% CMC-Na, dung mi gm cloroform-ethylacetate-isopropanol-

nc-amoniac t l 8 2 6 0,5 0,12 (theo th tch), pht hin cht phn tch ti 2

bc sng 254nm v 300nm. Phng php rt n gin, lch chun tng i

trong khong 0,73 - 0,98% v thu hi cao t 95,270% - 96,366%.

L.Wuqing v cng s [8] xc nh adenosine trong vin nang Jinshuibao.

Adenosine c tch trn bn mng silica GF254 CMC - Na2HPO4 vi dung mi

-9-

tng t nh trn, pht hin ti 2 bc sng l 260nm v 300nm. ng chun

c xy dng trong khong 0,65 - 5,2g vi r = 0,9995, thu hi ca phng

php l 99,5% v h s bin thin 3,7 %.

1.3.2. Phng php in di mao qun (CE) [6], [12]

Li Hai Ying v cng s [12] tch v nh lng adenosine, adenin, uridine

bng dung dch m dinatri tetraborat 15mM v natridihydrophosphat 14mM (pH =

9,5) vi 5% MeOH theo th tch, in th 18kV v detector UV bc sng 254nm.

H s tng quan ca phng php R 0,9985.

Feng-Qing Yang v cng s [6] tin hnh xc nh 12 nucleoside v

nucleobase (cytosine, adenine, guanine, cytidine, cordycepin, adenosine,

hypoxanthine, guanosine, inosine, 2-deoxyuridine, uridine v thymidine) trong mt

s loi ng trng h tho bng phng php in di mao qun khi ph (CE-MS).

Mu c ha tan bng H2O 950C, rung siu m nhit 75oC trong 30 pht. Sau

a dch chit v nhit phng, lc qua mng 0,25m. Dch chit c bm

vo h thng CE-MS vi pha ng l m formic 10mM cha 10% methanol, tc

dng 3L/pht. Phng php cho kt qu hm lng nucleoside v nucleobase

trong cordyceps sinensis l 9138 4823g/g cao hn so vi C.militaris l 3722

1446g/g.

1.3.3. Phng php sc k lng [9], [11], [17], [24], [25]

Lan -Fang Huang v cng s [9] tin hnh xc nh thnh phn hot tnh

ca Cordyceps sinensis v Cordyceps militaris gm adenin, hypoxanthin,

cordycepin v cc sn phm chuyn ha ca n bng phng php sc k lng khi

ph LC/ESI-MS. Phng php s dng pha ng gm H2O, methanol v acid

formic vi t l 85 : 14 : 1 (theo th tch), ct VP - ODS (2,0 x 150mm) ca hng

Shimadzu, la chn ion [M + H]+ ti m/z l 136, 137, 268, 252, 302 phn tch

nh lng cho 4 thnh phn hot tnh. ng chun c xy dng cho adenin vi

nng t 1,4 - 140g/mL, hypoxanthine t 0,6 - 117,5g/mL, cho adenosine t

0,5 - 128,5g/mL v cordycepin t 0,5 - 131,5g/mL. Gii hn pht hin v gii

hn nh lng ca adenin l 0,5 v 1,4g/mL, hypoxanthin l 0,2 v 0,6g/mL,

-10-

adenosine v cordycepin l 0,1 v 0,5g/mL. thu hi ca phng php trong

khong 93,7 - 107%.

Xiao Feng Xue v cng s [25] tin hnh phn tch adenosine trong sa ong

cha. Adenosine c chit bng ethanol : H2O t l 80 : 20 theo th tch. Dch

chit mu c bm vo h thng HPLC vi ct sc k C18 v dung mi gm 2

knh: knh A l acid phosphoric 0,4% v knh B l MeOH. Detector UV bc sng

257nm. thu hi ca phng php trong khong 91,6 - 98,3% vi lch chun

nh hn 5,3%. Gii hn pht hin v gii hn nh lng tng ng l 0,017 v

0,048g/mL.

Li chen v cng s [11] xc nh adenosine v cordycepin trong ng trng

h tho. Mu c chit vi H2O, sau dch chit c phn tch trn ct Eclipse

XDB -CN. Pha ng gm MeOH : H2O theo t l 7 : 93, s dng detector PDA ti

bc sng 260nm. Phng php c thu hi trong khong t 93,8 - 102,9%, vi

lch chun tng i nh hn 3,62%. Gii hn nh lng ca adenosine v

cordycepin l 0,21 v 0,083mg/lt. Phng php n gin, nhanh, chnh xc cao

v gi thnh thp.

Hemanth Kumar, M.Spandana [17] phn tch ng thi adenosine, cordycepin

v sn phm chuyn ha ca cordyceps sinensis bng phng php HPLC. iu

kin phn tch: ct C18 ProtoSIL (250nm x 4,6mm x 5m), pha ng MeOH : H2O

t l 20 : 80 theo th tch, tc dng 1mL/pht, detector UV bc sng 254nm,

nhit bung ct 300C. Hm lng ca adenosine v cordycepin khong 0,288 v

0,346mg/g. thu hi 101,1%, lch chun tng i l 1,33%.

Theo dc in Trung Quc [24], adenosine c chit tch t ngun

nguyn liu ng trng h tho nh sau: cn chnh xc 0,5g mu cho vo bnh cu,

thm 10mL MeOH 90%, y np, lc u, gia nhit v un hi lu trong 30 pht,

lm ngui, thm methanol 90% n lng chnh xc, lc u, lc. Chy sc k vi

iu kin nh sau: pha ng l m phosphat (pH = 6,5) v MeOH t l 17 : 3, ct

pha tnh octandecylsilane, pht hin ti bc sng 260nm.

-11-

1.4. Phng php sc k lng hiu nng cao (HPLC)

1.4.1. Nguyn tc chung ca sc k lng [2], [3], [4]

Sc k lng l qu trnh tch xy ra trn ct tch vi pha tnh l cht rn hoc

cht lng v pha ng l cht lng (sc k lng - rn, lng - lng). Mu phn tch

c chuyn ln ct tch di dng dung dch. Khi tin hnh chy sc k, cc cht

phn tch c phn b lin tc gia pha ng v pha tnh. Trong hn hp cc cht

phn tch, do cu trc phn t v tnh cht l ho ca cc cht khc nhau, nn kh

nng tng tc ca chng vi pha tnh v pha ng khc nhau, chng di chuyn vi

tc khc nhau v tch ra khi nhau.

1.4.1.1. Pha tnh trong sc k lng

Tu theo bn cht ca pha tnh, trong phng php sc k lng pha lin kt

thng chia lm 2 loi: sc k pha thng (NP-HPLC) v sc k pha o (RP-

HPLC).

+ Sc k pha thng: pha tnh c b mt l cc cht phn cc ( l cc silica

trn hoc cc silica c gn cc nhm ankyl c t cacbon mang cc nhm chc

phn cc: -NH2, -CN)

+ Sc k pha o: pha tnh thng l cc silica c ankyl ho, khng phn

cc, loi thng dng nht l C18H37

u im ca sc k pha o l tch v phn tch cc cht c phn cc rt a

dng: t rt phn cc, t phn cc ti khng phn cc. Hn na, trong rt nhiu

trng hp th thnh phn chnh ca pha ng li l nc nn rt kinh t.

1.4.1.2. Pha ng trong sc k lng

Pha ng trong sc k lng l thnh phn c cho i qua ct lin tc phn

tch cc hp cht trong mt hn hp, c nhng yu cu sau:

+ Pha ng phi tr vi pha tnh

+ Bn vng, n nh v khng b phn hu trong qu trnh chy sc k

+ Ho tan c mu

+ Phi c tinh khit cao, c nht thp v ph hp vi detector

C th chia pha ng lm hai loi:

-12-

+ Pha ng c phn cc cao: c thnh phn ch yu l nc, tuy nhin

phn tch cc cht hu c, cn thm cc dung mi gim phn cc. Pha ng

loi ny c dng trong sc k pha ngc.

+ Pha ng c phn cc thp: l cc dung mi t phn cc nh cyclopentan,

n-pentan, n-heptan, n-hexan, 2-chloropropan, cacbondisulfua (CS2), CCl4, toluene

tch mt nhm cht thng thng pha ng mt thnh phn i khi khng

p ng c kh nng ra gii, ngi ta thng phi hp 2 hay 3 dung mi c

c dung mi c phn cc t thp n cao ph hp vi php phn tch. S thay

i thnh phn pha ng i khi din ra theo thi gian, trng hp ny ngi ta gi

l gradient pha ng.

1.4.1.3. Detector trong sc k

Detector l b phn quan trng quyt nh nhy ca phng php. Tu thuc

bn cht l ho ca cht phn tch m la chn detector cho ph hp

+ Detector quang ph hp th phn t UV-Vis: p dng cho cc cht c kh

nng hp th nh sng trong vng t ngoi (UV) hoc vng kh kin (Vis).

+ Detector hunh quang: s dng pht hin cc cht c kh nng pht hunh

quang. i vi nhng cht khng c kh nng nh vy, cn phi dn xut ho cht

phn tch, gn n vi cht c kh nng pht hunh quang hoc cht phn tch phn

ng vi thuc th to thnh sn phm c kh nng pht hunh quang.

+ Detector dn: ph hp vi cc cht c hot tnh in ho: Cc ion, cc hp

cht c tnh dn in

+ Detector PDA: da vo s thay i cng bc x do s hp th bc x

ca hp cht c trong hn hp m chm tia bc x chiu qua. Da vo s thay i

cng bc x, xc nh c cht phn tch.

1.4.1.4. Cu to ca h thng sc k lng hiu nng cao

Cng nh tt c cc thit b sc k khc, thit b HPLC gm 3 phn chnh:

- Phn u vo cp pha ng c thnh phn mong mun v mu phn tch.

- Phn tch l phn trung gian ca h sc k bao gm ct tch, i khi c ct ph

tr.

-13-

- Phn pht hin v x l s liu gm cc detector, phn khuch i, computer v

phn mm x l s liu, b phn ghi tn hiu.

Hnh 1.5. S cu to h thng HPLC

1.4.2. Phn tch nh tnh v nh lng bng HPLC [2], [3]

Phn tch nh tnh: i lng c trng cho s tch sc k l thi gian lu

ca cc cht. Da vo thi gian lu ca cht phn tch trong chun v trong dung

dch phn tch nh tnh tng cht trong hn hp.

Hnh 1.6. Sc k trong phn tch sc k

nh lng cht phn tch, dng phng php ng chun, so snh hoc

thm chun.

* Phng php ng chun

- Pha ch mt s dung dch chun i chiu c nng khc nhau

- o p ng Si ca cc dung dch chun v lp ng cong ph thuc ca S

vo C:

S = f(C)

- Chn khong nng ph thuc tuyn tnh

- o tn hiu p ng Sx v ni suy nng Cx t ng chun

-14-

* Phng php so snh

- em o hp th quang ca dung dch chun

- o mt quang ca dung dch cn xc nh X

- Tnh nng ca dung dch X theo cng thc sau:

ch

chXX

D

CDC

.

* Phng php thm chun

- o cng p ng ca mu phn tch Sx.

- Pha ch mt s dung dch c nng Cx

- o nng cht chun i chiu CR khc nhau: Cx + CR, i

- o p ng ca cc dung dch Si

- Xy dng th s ph thuc Si = f(CR,i)

- Chn khong ph thuc tuyn tnh. Tnh nng Cx

-15-

Chng 2. I TNG V PHNG PHP NGHIN CU

2.1. i tng

i tng nghin cu: TPCN c cha THT

2.2. Ha cht v dng c

2.2.1. Thit b

- H thng sc k lng hiu nng cao ca Shimadzu

- My lc vortex VELP, my ng nht mu, my li tm lnh Hermle, my rung

siu m c ch gia nhit, cn phn tch (c chnh xc 0,1mg v 0,01mg), cn

k thut (c chnh xc 0,01g)

2.2.2. Dng c

- ng ly tm, ng ong, cc loi pipet, bnh nh mc

- ng nghim,....

2.2.3. Ha cht

Cc loi ha cht dng trong phng php u thuc loi tinh khit phn tch

* Chun adenososine t sigma aldrich tinh khit 99%

- Dung dch chun gc 1000g/mL: cn chnh xc khong 0,1000g adenosine

chun trn cn phn tch c chnh xc 10-4, ha tan v nh mc 100mL

bng H2O. Bo qun trong t lnh 40C, s dng trong 1 thng.

- Dung dch chun trung gian (200g/mL): ly 2mL dung dch chun gc vo

bnh nh mc 10mL v nh mc ti vch bng H2O.

- Cc dung dch chun lm vic nng 0,1; 0,5; 1,0; 5,0; 10,0; 20,0g/mL

c pha trong H2O.

* Cc loi ha cht, dung mi khc:

- Methanol (Merck 99,9%), n-hexan (Merck 99,9%), ethanol (Merck 99,9%),

diethyl ether (Merck 99,9%), ether du ha (Merck 99,9%), ethyl acetate

(Merck 99,9%)

- Nc ct dng cho sc k lng

-16-

2.3. Phng php nghin cu

2.3.1. Phng php ly mu

- i tng mu: TPCN c cha THT

- Phng php ly mu: ngu nhin

- a im ly mu: thnh ph H ni

- Khi lng mu ly: hai n v mu (hp, gi, l)/mu

- Bo qun v lu tr mu: iu kin thng

2.3.2. Phng php phn tch

Nguyn l: Adenosine c ha tan trong dung mi ph hp, tin hnh rung

siu m, ly tm. Dch chit c nh mc 50mL, lc v bm vo h thng sc k

lng hiu nng cao vi detector PDA.

* Quy trnh tm tt:

- Mu c ng nht k

- Cn mu vo ng ly tm, vi nn mu cha nhiu bo cn loi bo trc khi phn

tch

- Thm dung mi chit mu vo ng ly tm

- Lc vortex mu v dung mi c o trn u

- Rung siu m tng hiu sut ha tan cht phn tch vo dung mi chit

- ngui n nhit phng

- Ly tm vi tc cao, phn tch phn dung dch v lng phn b

- Chit lp mu vi dung mi trn

- Gp dch chit mu v nh mc n vch

- Lc

- Bm vo h thng HPLC

-17-

Hnh 2.1. Lc quy trnh phn tch mu d kin

2.4 Thm nh phng php phn tch [5]

Sau khi xy dng phng php phn tch cn phi c thm nh theo quy

nh ca USFDA, AOAC, USP cc thng s cn thm nh bao gm:

- Tnh c hiu, tnh chn lc (Specifility/Selectivity)

- Khong tuyn tnh v ng chun (Linearity and Calibration)

- Gii hn pht hin (Limit of Detection LOD)

- Gii hn nh lng (Limit of Quatification LOQ)

- ng (Trueness)

- chm (Precision)

- n nh ca phng php (Robustness/Ruggeness)

Dung mi chit

Chit lp

HPLC

nh mc

Siu m

Dch chit

Ly tm

Lc vortex

Loi bo (nu cn)

Lc

Cn mu

Cn

-18-

Vic la chn cc thng s thm nh ty thuc vo: K thut p dng, yu cu

ca phng php, iu kin v ngun lc ca phng th nghim. Trong phm vi v

thi gian nghin cu, chng ti thc hin thm nh phng php thng qua vic

nh gi cc thng s: Tnh c hiu, khong tuyn tnh v ng chun, gii hn

pht hin v gii hn nh lng, ng (thng qua hiu sut thu hi), chm

(thng qua lp li)

-19-

Chng 3. THC NGHIM, KT QU V BN LUN

3.1. La chn phng php phn tch

Chng ti tin hnh phn tch adenosine trn h thng sc k lng hiu nng

cao, detector PDA vi cc iu kin nh sau:

- Ct sc k C18 pha o

- Detector PDA ti bc sng 260 nm

- Tc dng: 1mL/pht

- Nhit bung ct: 40oC

- Th tch bm mu: 20L

Adenosine l hp cht c kh nng hp th ti bc sng trong vng t

ngoi nn c th s dng detector UV thay th. Tuy nhin, detector PDA c u

im l cho bit hnh dng ph ca cht phn tch. Khi kt hp gia thi gian lu v

hnh dng ph hp th c th khng nh chnh xc cht cn phn tch. Do , trong

nghin cu ny chng ti quyt nh s dng detector PDA.

Cht phn tch sau khi c tch khi ct sc k, c dn vo flowcell ca

u o v c chiu bi mt chm tia t ngoi. S hp th tia bc x bi cc cht

tan tun theo nh lut Lambert-Beer.

Hnh 3.1. Ph hp th ca adenosine

3.2. Ti u iu kin xc nh adenosine trn h thng HPLC

3.2.1. La chn ct tch sc k

Ct tch c vai tr quan trng trong vic tch cc cht phn tch ra khi

nhau, n c v nh tri tim ca h thng sc k. Hin nay, sc k pha o c

-20-

s dng rng ri, do tnh ph bin, tnh kinh t v c hiu qu cao. Do , chng ti

la chn h sc k pha o, kho st kh nng tch ca cht phn tch trn mt s

ct C18 v c nh cc iu kin phn tch khc vi nng adenosine 10,5ppm:

- Ct X-Bridge C18 (150mm 2,1mm 3,5m)

- Ct Symmetry C18- Waters (150mm 4,6mm 5m)

- Ct IntertSustain C18 (250mm 4,6mm 5m)

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min

0

25

50

mAU260nm4nm (1.00)

/3.3

03

/73

50

82

Hnh 3.2. Sc phn tch trn ct X-Brigde C18

Hnh 3.3. Sc phn tch trn ct Symmetry C18

-21-

0.0 2.5 5.0 7.5 10.0 12.5 min

0

25

50

75

100

125mAU

260nm,4nm (1.00)

/10

.36

1/7

52

30

6

Hnh 3.4. Sc phn tch trn ct IntertSustain C18

Vi ct X-Brigde C18, thi gian lu ngn (khong 3,5 pht), do khi phn

tch trn nn mu thc s b nh hng bi tp cht do khng tch c cht phn

tch v tp cht. Hn na, ct X-Brigde C18 (150mm x 2,1mm x 3,5m) c kch

thc ht nh nn p sut ca ct cao c hi cho bm v h thng sc k. Nu hot

ng lin tc p sut cao nh vy, bm d xy ra hin tng p sut khng n

nh dn n tc dng khng n nh.

Khi s dng ct Symmetry C18 (150mm 4,6mm 5m) hnh dng peak t,

chn peak dong, thi gian lu ngn (khong 6,1 pht). Nh vy, khi phn tch trn

nn mu thc c th cht phn tch b ln peak tp hoc s b dnh peak.

Khi s dng ct IntertSustain C18 (250mm 4,6mm 5m) tn hiu peak

nhn, cn i, rng chn peak nh khong 0,2 pht, thi gian lu 10,3 pht. V

vy khi phn tch trn nn mu thc, cht phn tch c th c tch tt, trnh c

hin tng chng peak, dnh peak vi cc tp cht khc. Do , trong nghin cu

chng ti s dng ct IntertSustain C18 (250mm 4,6mm 5m) tch v nh

lng adenosine ng thi tin hnh cc kho st tip theo.

3.2.2. Kho st thnh phn pha ng

Sau pha tnh th pha ng l yu t th hai quyt nh n hiu qu tch sc

k. Nhn chung, pha ng c th nh hng n chn lc ca h pha, thi gian

lu gi, hiu lc ca ct tch, phn gii, rng ca peak sc k...

-22-

Chng ti tin hnh nghin cu cc pha ng khc nhau gm: Knh A l

H2O, knh B l dung mi ACN hay MeOH, c nh cc iu kin phn tch khc.

Qu trnh kho st cho kt qu nh sau:

Bng 3.1. Kho st thnh phn pha ng

Thnh phn pha ng Thi gian

lu (pht) Kt qu

Knh A Knh B

H2O ACN 9,5

Peak dong, rng chn peak ln, tn

hiu cht phn tch thp, tn hiu nhiu

ng nn cao hn.

H2O MeOH 10,3

Peak rt nhn, cn i, rng chn

peak nh, tn hiu cht phn tch cao

hn, tn hiu nhiu ng nn rt thp.

Hnh 3.5. Sc phn tch s dng pha ng H2O v ACN

0.0 2.5 5.0 7.5 10.0 12.5 min

0

25

50

75

100

125mAU

260nm,4nm (1.00)

/10

.34

1/7

51

16

9

Hnh 3.6. Sc phn tch s dng pha ng H2O v MeOH

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 min

-5

0

5

10

15

20

25

30

35

mAU260nm,4nm (1.00)

/9.5

40

/72

19

87

-23-

T kt qu trn, chng ti nhn thy:

C th s dng c 2 dung mi l MeOH v ACN tch v nh lng

adenosine. Tuy nhin, khi s dng MeOH tn hiu peak rt nhn, cn i, nhy

cao, nhiu ng nn thp hn so vi dung mi ACN. Hn na, gi tr kinh t

mang li khi s dng dung mi MeOH ln hn rt nhiu so vi dung mi l ACN.

V vy, trong nghin cu ny chng ti s dng MeOH lm thnh phn pha ng

phn tch adenosine.

3.2.3. Kho st chng trnh ra gii gradient

Trong thnh phn cu to ca ng trng h tho c cha rt nhiu cc cht

thuc nhm nucleoside c tnh cht tng t nhau, nu ch ra gii cht phn tch

theo chng trnh c nh pha ng, cc cht trong nhm c th c cng thi gian

lu dn n hin tng chng peak hoc dnh peak. Do vy, tng nhy ca tn

hiu peak, tng kh nng tch sc k, trnh hin tng dong peak, chng peak,

dnh peak, chng ti kho st chng trnh gradient tch v nh lng

adenosine trong ng trng h tho v c nh cc thng s khc.

Kt qu thu c khi chy chng trnh gradient nh sau:

Gradient 1

Thnh phn pha ng % Thi gian lu

(pht)

Knh A = H2O Knh B = MeOH 13,2

92 8

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

0

5

10

15

20mAU

260nm4nm (1.00)

/13

.20

4/7

41

70

4

Hnh 3.7. Sc phn tch theo chng trnh gradient 1

-24-

Gradient 2

Thnh phn pha ng % Thi gian lu

(pht) Knh A = H2O Knh B = MeOH

80 20 9,5

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 min

-5

0

5

10

15

20

25

30

35

mAU260nm,4nm (1.00)

/9.5

40/7

2198

7


Gradient 3

Thi gian

(pht) Thnh phn pha ng (%)

Thi gian

lu (pht)

Knh A = H2O Knh B = MeOH

0,01 100 0

15,2

2 95 5

8 80 20

11 80 20

12 100 0

16 100 0

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

0

10

20

30

40mAU

260nm4nm (1.00)

/15

.28

9/3

56

90

4


-25-

Gradient 4

Thi gian (pht) Thnh phn pha ng (%) Thi gian lu


0,01 100 0

12,9

2 95 5

5 80 20

10 90 10

12 95 5

15 100 0

0.0 2.5 5.0 7.5 10.0 12.5 min

0

25

50

mAU260nm,4nm (1.00)

/12

.99

6/7

15

97

6


Gradient 5

Thi gian

(pht)

Thnh phn pha ng (%) Thi gian lu


0,01 100 0

10,3

2 85 15

5 70 30

10 55 45

12 85 15

15 100 0

-26-

0.0 2.5 5.0 7.5 10.0 12.5 min

0

25

50

75

100

125mAU

260nm,4nm (1.00)

/10

.36

1/7

52

30

6


Khi s dng thnh phn pha ng gm H2O v MeOH theo chng trnh

gradient 1 thi gian lu di (13,2 pht) v gim cn 9,5 pht khi tng t l MeOH

ln 20% chng trnh gradient 2. Tuy nhin, c 2 chng trnh peak u dong,

rng chn peak ln. V vy, khi phn tch hiu qu tch sc k khng tt.

Nu s dng chng trnh gradient 3 v 4 thi gian lu ca cht phn tch rt

mun (12,9 - 15,2 pht) dn n tn dung mi, ko di thi gian phn tch, nh

hng n hiu qu kinh t. Hn na, tn hiu cht phn tch thp lm gim LOQ

ca phng php. Khi tng t l MeOH (theo chng trnh gradient 5), thi gian

lu gim v nhy ca cht phn tch ln hn, peak nhn, cn i hn. Do ,

chng ti s dng chng trnh 5 tch v nh lng cht phn tch.

3.2.4. iu kin ti u ca qu trnh sc k

Sau khi kho st, chng ti la chn c iu kin ti u cho qu trnh

sc k nh sau :

- Ct IntertSustain C18 (250mm x 4,6mm x 5m)

- Detector PDA bc sng 260 nm

- Pha ng gm 2 knh: knh A l H2O v knh B l MeOH theo chng trnh

ra gii gradient 5

- Tc dng: 1mL/pht

- Nhit ct: 40oC

- Th tch bm mu: 20L

-27-

0.0 2.5 5.0 7.5 10.0 12.5 min

0

25

50

75

100

125mAU

260nm,4nm (1.00)

/10

.36

1/7

52

30

6

Hnh 3.12. Sc phn tch vi iu kin ti u

3.3. Kho st quy trnh x l mu

Quy trnh x l mu d kin theo hnh 2.1. Theo quy trnh d kin ny chng

ti tin hnh kho st mt s yu t nh hng ti qu trnh chit nh: dung mi

chit mu, dung mi loi bo, nhit chit, thi gian chit, s ln chit mu trn

nn mu TPCN c cha THT.

3.3.1. Kho st dung mi chit

Chng ti s dng dung mi chit l hn hp H2O - MeOH v hn hp H2O

- EtOH vi cc t l khc nhau la chn c dung mi chit ph hp nht.

Trn cng mt nn mu TPCN c cha THT chng ti tin hnh chit theo

lc d kin. S dng dung mi trn vi cc t l khc nhau. Vi mi loi dung

mi phn tch lp li 2 ln, tnh kt qu trung bnh. Kt qu phn tch c ch ra

trong hnh sau:

Hnh 3.13. Kho st dung mi chit

-28-

Khi s dng dung mi l EtOH hoc t l gia H2O : EtOH = 9 : 1 hm

lng adenosine thu c trong nn mu phn tch rt thp (khong 12,9 -

28,5mg/100g). Trong khi , vi dung mi chit MeOH, hiu lc chit tt hn so

vi EtOH, hm lng adenosine thu c trong mu l 34,7mg/100g. Tuy nhin,

khi tip tc gim t l MeOH v tng t l H2O hm lng adenosine tng ln ng

k v t hiu lc chit cao khi s t l H2O : MeOH = 9 : 1 (65,5mg/100g) v thay

i khng ng k khi dung mi l 100% H2O (65,6mg/100g). Tuy nhin, trong nn

mu thc phm chc nng rt a dng v phc tp, khi s dng dung mi H2O c

rt nhiu cht c kh nng ha tan khin cho dung dch chit thu c ln tp nhiu,

khi phn tch gy bn ct v h thng sc k. Trong khi , vi t l H2O : MeOH =

9:1 s hn ch c s ha tan ca mt s cht lm cho dch chit mu sch, m

bo an ton cho ct v h thng khi phn tch. Do , chng ti s dng hn hp

H2O : MeOH theo t l 9 : 1 chit adenosine ra khi nn mu phn tch.

3.3.2. Kho st dung mi loi bo

i vi cc sn phm TPCN, nu thnh phn bo khng c loi b s nh

hng n kh nng ha tan ca cht phn tch trong dung mi chit. Hn na, dch

chit mu bn khi phn tch trn h thng sc k d gy bn ct v h thng, sau

mt thi gian phn tch, p sut ct tng, hnh dng peak xu, tui th ct ngn.

ng thi, tp nhiu cng lm gim tnh chn lc i vi cc cht phn tch. V

vy, chng ti kho st mt s dung mi hu c khng phn cc gm: n-hexan,

diethyl ether, ether du ha loi bo trong cc TPCN.

Trn nn mu TPCN c cha THT dng vin nang v dng dch lng c

cha nhiu bo, tin hnh so snh gia nn mu khng loi bo v nn mu c loi

bo bng cc dung mi trn. S dng 20mL dung mi mi loi, tin hnh lc xoy,

rung siu m, ly tm, thnh phn bo s c ha tan trong cc dung mi hu c v

tch lp trn, dng pipet pasteur ht phn dch trn, sau tin hnh chit theo

quy trnh d kin, dch chit sau c phn tch trn h thng sc k. Kt qu

trong dch chit ca mu khng loi bo rt c, khi phn tch trn h thng HPLC,

peak sc k c cha nhiu tp cht nh hng n ct sc k v h thng. Hn na,

-29-

n lm gim kh nng ha tan ca cht phn tch trong dung mi chit. Do , lm

gim hiu sut chit.

Khi s dng dung mi diethyl ether hay ether du ha, s phn lp gia 2

pha rt kh quan st, kh nng loi bo km, hm lng adenosine thu c thp.

Nu s dng dung mi loi bo l n-hexan dch chit mu thu c rt trong, kh

nng tch gia 2 pha r rng nn vic loi bo thun tin hn. Mt khc, hm lng

adenosine thu c khi s dng dung mi ny l ln nht. V vy, chng ti la

chn n-hexan loi bo trong mu.

3.3.3. Kho st nhit chit

Trn cng mt nn mu TPCN c cha THT, chng ti tin hnh chit

cc nhit khc nhau: nhit thng, 45oC, 60oC, 75oC, 100oC kt hp vi rung

siu m. mi mc nhit tin hnh lm lp 2 ln, ly kt qu trung bnh.

Kt qu kho st thu c nh sau:

Hnh 3.14. Kho st nhit chit

Nh vy, hm lng adenosine thu c khi chit tng dn theo nhit , t

hm lng cao nht nhit 100oC l 60,8mg/100g. Do khi nhit tng, ha

tan ca cht phn tch trong dung mi chit mu tng; ng thi thc y qu trnh

chuyn ha t AMP thnh adenosine. V vy, hm lng adenosine thu c ln.

Do , trong nghin cu ny chng ti s dng nhit 100oC chit cht phn

tch ra khi nn mu.

-30-

3.3.4. Kho st thi gian chit

Trn nn mu TPCN, chng ti tin hnh chit mu theo quy trnh d kin

vi thi gian thay i: 15 pht, 30 pht, 60 pht, 90 pht v 120 pht, s dng cc

iu kin c ti u, kt qu thu c nh sau:

Hnh 3.15. Kho st thi gian chit

Hm lng adenosine thu c trong mu tng khi thi gian chit mu tng

t 5 pht n 30 pht. Tuy nhin, nu tip tc tng thi gian chit ln 60 pht, 90

pht, 120 pht th hm lng adenosine gim dn do s chuyn ha ca adenosine

sang adenin hoc inosin trong qu trnh trao i cht. Do , chng ti la chn

thi gian 30 pht chit adenosine ra khi nn mu.

3.3.5. Kho st s ln chit

Trn cng mu TPCN c cha THT chng ti tin hnh chit lp 4 ln

kho st kh nng chit. Ti mi ln chit chng ti lm lp 2 ln song song, ly kt

qu trung bnh. Kt qu thu c nh sau

Bng 3.2. Kho st s ln chit

S ln chit (ln) 1 2 3 4

Hm lng adenosine (mg/100g) 43,7 69,0 69,3 70,1

Kt qu phn tch cho thy: hm lng ca adenosine tng theo s ln

chit. Tuy nhin, ln chit 3 v 4 hm lng tng khng ng k so vi ln 2. V

vy chng ti tin hnh chit lp 2 ln vn m bo hiu sut v tit kim dung mi.

-31-

Sau khi kho st, chng ti quyt nh s dng phng php chit adenosine

t TPCN theo quy trnh c trnh by sau y:

Hnh 3.16. Quy trnh phn tch mu thc

30mL H2O:MeOH = 9:1

Chit lp ln 2

HPLC

nh mc

Siu m (1000C, 15 pht)

Gn dch chit vo bnh nh mc 50 mL

Ly tm ngui (6000rpm, 5 pht)

Lc vortex (10 pht)

Loi bo (nu cn)

Lc

Cn, ong mu (cn 0,1-0,5g mu rn hay ly 5mL mu lng) vo ng ly tm 50mL

Cn

20mL n-hexan

-32-

3.4. nh gi phng php phn tch

3.4.1. Kho st khong tuyn tnh v lp ng chun

Khong nng cht phn tch ph thuc tuyn tnh vo chiu cao hay din

tch peak gi l khong tuyn tnh. Mi quan h tuyn tnh gia nng cht phn

tch vi tn hiu o c gi l ng chun. Ch tiu u tin ca mt ng

chun t yu cu l h s tng quan hi quy R phi t theo yu cu sau:

0,995 R 1 hay 0,99 R2 1

Vi cc iu kin chy HPLC chn, chng ti tin hnh kho st s ph

thuc tuyn tnh ca din tch peak sc k vo nng ca adenosine. T dung dch

chun gc, tin hnh pha long thnh dung dch chun trung gian v dy chun lm

vic cc nng khc nhau. Sau tin hnh bm vo h thng HPLC, thu c

kt qu nh sau:

Bng 3.3 . Kho st khong tuyn tnh

Nng (g/mL) Din tch (mAU) Nng (g/mL) Din tch (mAU)

350 17920193 10,5 744575

263 16570462 5,03 371489

210 15379030 2,10 149170

189 13843488 1,05 74495

105 7135000 0,500 37186

50,3 3616964 0,210 15565

21,0 1493088 0,105 7771

0

2000000

4000000

6000000

8000000

10000000

12000000

14000000

16000000

18000000

20000000

0 100 200 300 400

Hnh 3.17. Kho st khong tuyn tnh

-33-

T th cho thy, khi tng dn nng th din tch peak cng tng, th

tuyn tnh trong khong t 0,105g/mL n 210g/mL. Nh vy, khong tuyn tnh

ca adenosine t 0,105 - 210g/mL.

Tin hnh xy dng ng chun: Trong khong tuyn tnh va xc nh,

thit lp phng trnh tng quan gia nng v din tch peak theo phng trnh

hi quy tuyn tnh, thu c phng trnh ng chun:

Y = 72871X - 47211 vi R2 = 0,9992

Hnh 3.18. ng chun ca adenosine

3.4.2. Gii hn pht hin (LOD) v gii hn nh lng (LOQ) ca phng php

Gii hn pht hin (LOD): l nng thp nht ca cht phn tch c th xc

nh c nhng khng nht thit phi nh lng c trong iu kin th nghim

c th. Trong sc k, LOD c th c xc nh l nng ca cht phn tch m

ti t l gia tn hiu ca cht phn tch v tn hiu nn bng 3 (S/N =3).

Gii hn nh lng (LOQ): l nng ti thiu ca mt cht c trong mu

th m ti c th nh lng c bng phng php phn tch v cho kt qu c

chnh xc mong mun. LOQ c th c xc nh l nng ca cht phn tch

m ti t l gia tn hiu ca cht phn tch v tn hiu nn bng 10 (S/N =10).

Do , c th tnh LOQ t LOD theo cng thc: LODLOQ3

10

-34-

Cch tin hnh: S dng nn mu thc (TPCN c cha THT dng vin

nang, dng dung dch) tin hnh pha long v bm vo h thng sc k cho ti khi

tn hiu peak sc k gp 3 ln tn hiu nhiu ng nn. T xc nh c LOD

v LOQ nh sau:

Bng 3.4. LOD v LOQ ca adenosine

Nn mu LOD (g/kg, g/lt) LOQ (g/kg, g/lt)

Dng vin nang 20 66,67

Dng dung dch 14 46,67

Hnh 3.19. Sc nn mu TPCN dng vin nang ti LOD

Hnh 3.20. Sc nn mu TPCN dng lng ti LOD

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

-0.5

0.0

0.5

1.0

mAU260nm4nm (1.00)

/9.8

94

/39

/10

.38

3/1

21

-35-

3.4.3. nh gi c hiu ca phng php

Tnh c hiu: l kh nng pht hin c cht phn tch khi c mt cc tp

cht khc nh tin cht, cc cht chuyn ha, cc cht tng t tp cht C th,

trong php phn tch nh lng, l kh nng xc nh chnh xc cht phn tch

trong mu khi b nh hng ca tt c cc yu t khc.

Khi s dng detector PDA, tnh c hiu ca phng php c c trng

qua tinh khit ca peak (peak purity). Trn sc phn tch chun v thc chng

ti tin hnh xc nh c hiu ca phng php thu c kt qu nh sau:

Hnh 3.21. c hiu ca chun adenosine

Hnh 3.22. c hiu ca phng php trn nn mu dng nang cng

-36-

Hnh 3.23. c hiu ca phng php trn nn mu dng nang mm

Hnh 3.24. c hiu ca phng php trn nn mu dng lng

Kt qu cho thy, tinh khit ca peak trong cc trng hp u ln hn

0,999, chng t phng php c c hiu tt.

3.4.4. nh gi lp li ca h thng

lp li ( chm) th hin s gn nhau ca cc kt qu o so vi gi tr

trung bnh, l mc thng nht ca cc kt qu th ring bit khi quy trnh phn

tch c p dng lp li trn cng mt mu. lp li c th hin bng lch

chun tng i RSD%. S dng dung dch chun 10,5g/mL tin hnh tim lp 10

ln vo h thng HPLC, tnh lp li ca thi gian lu v din tch peak.

Bng 3.5. Kho st lp li ca h thng

Ln Thi gian lu (pht) Din tch peak (mAU)

1 10,321 746099

2 10,603 752840

3 10,361 752306

4 10,341 751169

-37-

Ln Thi gian lu (pht) Din tch peak (mAU)

5 10,335 753232

6 10,324 753154

7 10,322 752909

8 10,332 752767

9 10,328 753088

10 10,332 753030

Trung bnh 10,360 752059

RSD % 0,83 0,29

Kt qu cho thy lp li ca thit b rt tt, lch chun tng i ca

thi gian lu l 0,83% v din tch peak l 0,29% - u nm trong gii hn cho

php ca AOAC. Nh vy, khi phn tch adenosine h thng cho cc kt qu rt n

nh.

3.4.5. lp li v thu hi ca phng php

y l hai yu t quan trng nh gi hiu qu ca phng php phn tch.

Mt phng php phn tch tt ngoi vic c sai s nh cn yu cu c lp li

cao. Theo l thuyt thng k, cc i lng c trng cho lp li l lch

chun SD v lch chun tng i RSD%.

thu hi c tin hnh xc nh da trn k thut thm chun. thu hi

R% c tnh nh sau:

%100)(

%

c

mcm

C

CCR

Trong : Cm+c: Nng ca mu thm chun

Cm: Nng ca mu

Cc: Nng ca chun thm vo mu

Cch tin hnh: Trn nn mu thc chng ti tin hnh phn tch lp li 6 ln

v thm chun 3 mc nng khc nhau (mc thp, trung bnh v mc cao), sau

tin hnh phn tch lp 3 ln. Kt qu phn tch c ch ra trong cc bng sau:

-38-

Bng 3.6. lp li v thu hi trn nn mu dng vin nang mm

Mu thc m (g) mc (mg) mm + c (mg/g) TB SD RSD% R%

1 0,4001

0

0,3991

0,4099 0,0108 2,65

2 0,4102 0,4172

3 0,4608 0,4082

4 0,4045 0,3955

5 0,4103 0,4222

6 0,4505 0,4174

1a 0,3828

0,0525

0,5359

0,5220 0,0168 3,22 96,99 1b 0,3561 0,5268

1c 0,4826 0,5033

2a 0,4093

0,105

0,6499

0,6588 0,0123 1,87 95,30 2b 0,3983 0,6537

2c 0,3526 0,6729

3a 0,3829

0,210

0,9572

0,9408 0,0295 3,13 104,80 3b 0,3808 0,9585

3c 0,4282 0,9068

Bng 3.7. lp li v thu hi trn nn mu dng vin nang cng

Mu thc m (g) mc

(mg) mm + c (mg/g) TB SD RSD% R%

1 0,3716

0

0,3048

0,3021 0,0080 2,64

2 0,3526 0,3003

3 0,4278 0,2898

4 0,3423 0,3056

5 0,3536 0,3135

6 0,4102 0,2984

1a 0,3762

0,0525

0,4365

0,4434 0,0203 4,58 100,13 1b 0,3743 0,4274

1c 0,3895 0,4662

-39-

Mu thc m (g) mc

(mg) mm + c (mg/g) TB SD RSD% R%

2a 0,3332

0,105

0,5981

0,5861 0,0228 3,89 96,22 2b 0,3789 0,5598

2c 0,3283 0,6003

3a 0,3913

0,210

0,8024

0,8516 0,0443 5,20 96,24 3b 0,3522 0,8640

3c 0,3396 0,8884

Bng 3.8. lp li v thu hi trn nn mu dng lng

Mu thc V (mL) mc

(mg) mm + c (mg) TB SD RSD% R%

1 5

0

0,0796

0,0784 0,0028 3,56

2 5 0,0801

3 5 0,0746

4 5 0,0750

5 5 0,0806

6 5 0,0803

1a 5

0,0315

0,1094

0,1055 0,0034 3,19 85,95 1b 5 0,1037

1c 5 0,1034

2a 5

0,063

0,1331

0,1329 0,0015 1,11 86,45 2b 5 0,1342

2c 5 0,1313

3a 5

0,126

0,1969

0,2003 0,0037 1,86 96,72 3b 5 0,1997

3c 5 0,2042

-40-

Nh vy, lch chun tng i ca adenosine trong cc nn mu thc

phm chc nng nm trong khong 1,11% - 5,20% v thu hi ca phng php

khong 85,95% - 104,80%. Nh vy, kt qu t yu cu ca AOAC, chng t

phng php c lp li v thu hi ph hp.

3.5. nh gi cht lng ca TPCN c cha THT

Chng ti tin hnh phn tch hm lng adenosine ca mt s sn phm

TPCN c cha THT. T , nh gi cht lng ca mt s sn phm TPCN c

cha THT lu thng trn th trng H Ni.

Tin hnh thu thp ngu nhin mt s sn phm chc nng ng trng h

tho hin ang lu thng trn th trng H Ni. Sau tin hnh phn tch hm

lng adenosine theo quy trnh kho st. So snh kt qu phn tch c ti

phng th nghim vi kt qu cng b trn nhn ca sn phm. T nh gi cht

lng ca cc sn phm ny. Kt qu phn tch c ch ra nh sau:

Hnh 3.25. nh gi cht lng mu TPCN c cha THT

Thc nghim cho thy c 57,1% mu c hm lng adenosine t hm

lng ghi nhn, s sn phm khng t l 28,5%, s sn phm cn li khng c

cng b (14,3%).

Mt s sn phm ny ch cng b hm lng cordyceps b sung vo m

khng cng b c th hm lng adenosine, mc d trong cc loi cordyceps khc

nhau hm lng cc nucleosides khc nhau. C th, trong cordyceps sinensis hm

-41-

lng adenosine cao v hm lng cordycepin thp. Ngc li vi cordyceps

militaris hm lng cordycepin l ch yu. V vy, khuyn co cc nh sn xut

nn cng b c th ngun nguyn liu b sung l cordyceps sinensis hay cordyceps

militaris vo sn phm c th nh gi cht lng thc ca sn phm.

3.6. Bn lun

3.6.1. i tng nghin cu

ng trng h tho l mt dc liu qu, c gi tr cao, do vic tiu

chun ha cht lng ca chng l rt quan trng. Dc in Vit Nam cha cp

n dc liu ny v cc ch phm ca n, v vy gp phn qun l cht lng

cho cc sn phm c cha ng trng h tho chng ti la chn ti ny

nghin cu.

Tuy nhin, do iu kin v thi gian nn chng ti mi ch thc hin c

vi trn 20 mu thc t v ly mu ngu nhin.

3.6.2. Xy dng quy trnh phn tch

Adenosine l hp cht c kh nng hp th ti bc sng trong vng t

ngoi nn c th s dng detector UV phn tch. Tuy nhin, detector PDA c u

im l cho bit hnh dng ph ca cht phn tch. Khi kt hp gia thi gian lu v

hnh dng ph hp th c th khng nh chnh xc cht cn phn tch. Do , trong

nghin cu ny chng ti quyt nh s dng detector PDA.

Kt qu ca ti ny chng ti tm c cc iu kin ti u cho qu

trnh phn tch trn h thng HPLC kt ni detector PDA vi h dung mi thng

dng, r tin, ct tch thng dng.

3.6.3. Kho st quy trnh x l mu

Chng ti kho st v tm c cc iu kin chit adenosin t mu thc

phm chc nng cho hiu sut chit cao v lp tt. Thi gian chit tng i

ngn, tn t dung mi.

i vi cc mu c cha nhiu bo nh dng vin nang, ch phm cn c

loi bo trc khi tin hnh x l nh cc mu khc.

-42-

3.6.4. Thm nh phng php

Sau khi xy dng quy trnh phn tch, chng ti tin hnh thm nh phng

php theo AOAC. Cc tiu ch thm nh u t theo yu cu ca AOAC, khong

tuyn tnh ca phng php rng, cho php nh lng adenosin trong cc i

tng a dng, hn ch sai s khi phi lm giu mu hoc pha long mu.

3.6.5. Kt qu phn tch mu thc

Do cha c tiu chun cho cc sn phm ca ng trng h tho nn trong

nghin cu ny chng ti tm thi chp nhn ch phm t yu cu khi hm lng

nm trong khong 90 110% so vi hm lng ghi nhn. Vi iu kin , c

57,1% ch phm t yu cu v hm lng.

Nh vy, mt lng khng nh sn phm khng t yu cy hoc khng

cng b hm lng, v vy chng ti cho rng cn phi kim sot cht ch cc sn

phm thc phm chc nng hn na.

-43-

KT LUN V KIN NGH

Trn c s cc kt qu thc nghim nghin cu xc nh hm lng

adenosine trong TPCN c cha THT bng phng php sc k lng hiu nng cao

kt ni detector PDA, chng ti thu c cc kt qu sau:

1. Ti u ha c cc iu kin chy sc k lng hiu nng cao cho xc nh

adenosine trong TPCN bao gm:

- Kho st v tm c cc iu kin ti u cho phn tch sc k: ct sc k

Intersustain C18 (250mm 4,6mm 5m); pha ng gm 2 knh: knh A l H2O,

knh B l MeOH; chng trnh ra gii gradient, detector PDA bc sng 260nm.

- Xy dng c quy trnh chit mu: Mu c loi bo bng n-hexan (nu

mu cha bo). Sau , chit trong hn hp H2O : MeOH t l 9 : 1, s dng my

rung siu m c ci t nhit 100oC, chit lp 2 ln, vi tng thi gian 30 pht.

2. Xy dng c ng chun t 0,105 - 210g/mL, phng php c gii

hn pht hin 14 - 20g/kg hoc g/L v gii hn nh lng tng ng t 46,67

66,67g/kg hoc g/L ty tng nn mu, lch chun tng i t 1,11 5,20%

v thu hi 85,95 104,80%.

3. phn tch trn 20 mu TPCN c cha THT ang lu thng trn th

trng H Ni. Kt qu phn tch cho thy c 57,1% mu c hm lng adenosine

t so vi cng b trn nhn. Cc sn phm cn li khng t hoc khng cng b

c th hm lng cht chnh ny.

T cc kt qu thu c, chng ti nhn thy phng php phn tch n

gin, c nhy t yu cu, phn tch nhanh v chnh xc, c th p dng kim

nghim rng ri ti cc phng th nghim c thit b HPLC vi tin cy cao.

Trong tng lai, chng ti s tip tc m rng phng php phn tch ng

thi nhiu cht khc thuc nhm nucleosides. V tip tc m rng i tng phn

tch, khng ch trong phm vi TPCN m trn cc i tng sn phm khc.

TI LIU THAM KHO

Ting Vit

1. B Y t (2012), Dc l hc tp 2, Nh xut bn Y hc, H ni.

2. Trn T Hiu, T Vng Nghi, Nguyn Vn Ri, Nguyn Xun Trung (2007),

Ha hc phn tch phn 2, Nh xut bn Khoa hc v k thut, H ni.

3. Cao Minh Quang (2006), Sc k lng hiu nng cao, Nh xut bn Y hc,

Thnh ph H Ch Minh.

4. Nguyn Vn Ri (2009), Gio trnh Cc phng php tch, khoa Ha hc

trng i hc Khoa hc T nhin, i hc Quc gia H ni.

5. Vin Kim nghim An ton V sinh thc phm quc gia (2010), Thm nh

phng php trong phn tch ha hc v vi sinh, Nh xut bn Khoa hc v k

thut, H ni.

Ting Anh

6. Feng-Qing Yang, Liya Ge, Jean Wan Hong Yong, Swee Ngin Tan, Shao-

Ping Li (2009),"Determination of nucleosides and nucleobases in different species

if Cordyceps by capillary electrophoresis-mass spectrometry", Journal of

Pharmaceutical and Biomedical Analysis, 50, 307-314.

7. J. Zhao, J. Xie, L.Y. Wang, S.P. Li (2013), "Advanced development in

chemical analysis of Codyceps", Journal of Pharmaceutical and Biomedical

Analysis.

8. L. Wuqing, Long Xinhua, et al (1996), "Determination of adenosin in

Jinshuibao capsule by TLC scanning", Chinese Traditional and Herbal Drugs, 1.

9. Lan-Fang Huang, Yi-Zeng Liang, Fang-Qui Guo, Zhi-Feng Zhou, Ben-Mei

Cheng (2003), "Simultaneous separation and determination of active components

in Cordyceps sinensis and Cordyceps militarris by LC/ESI-MS", Journal of

Pharmaceutical and Biomedical Analysis, 33, 1155-1162.

10. Lei Huang, Qizahang Li, Yiyuan Chen, Xuefei Wang and Xuanwei Zhou

(2009), "Determination and analysis of cordycepin and adenosine in the products

of Cordyceps spp.", African Journal of Microbiology Research, Vol. 3, 957-961.

11. Li Chen, Yan Aiguo, Cai Chunyan, Liu Zhiping (2012), "Fast

determination of adenosine and cordycepin in Cordyceps and its deserted solid

medium", Chinese Journal of Chromatography,Vol. 30 Issue (07), 711-715

12. Li Hai Ying, Yang Geng Liang, Wang De Xian, Liu Hai Yan, Li Bao Hui

(2001), "Determination of the active components in artificial cordyceps by

capillary zone electrophoresis", Journal of Hebei University (Natural Science

Edition).

13. Lucia Spicuzza, Giuseppe Di Maria, Riccardo Polosa (2006), "Adenosine

in the airways: Implications and applications", European Journal of

Pharmacology, 533, 77-88.

14. M.G. Shashidhar, P. Giridhar, K. Udaya Sankar, B. Manohar (2013),

"Bioactive principles from Cordyceps sinensis: A potent food supplement - A

review", Journal of Functional foods.

15. Ma Yan, Wang Yu, Yang Guang-zhao, Yu Rong-min (2008),

"Determination of nucleosides in Cordyceps sinensis preparation by dual-

wavelength TLC-scanning", China Pharmacy, 30.

16. Mizuno T. (1999), "Medicinal effects and utilization of Cordyceps (Fr.)

Link (Ascomycetes) and Isaria Fr. (Mitosporicfungi) Chinese caterpillar fungi",

International Journal of Medicinal Mushrooms, 1, 251-262.

17. Mr. Hemanth Kumar, M. Spandana (2013), Simultaneous extraction,

determination and analysis of adenosine, cordycepin and other derivatives of

Cordyceps sinensis of Nepal by new validated HPLC method, Journal of

Pharmacognosy and Phytochemistry, Volume 2, Issue 4.

18. Nakamura K., Konoha K., Yamaguchi Y., Kagota S., Shinozuka K.,

Kunitomo M. (2003), "Combined effects of Cordyceps sinensis and methotrexate

on hematogenic lung metastasis in mice", Recept. Chan, 9, 329-334.

19. Nakamura K., Yamaguchi Y., Kagota S., Kwon Y.M., Shinozuka K.,

Kunitomo M. (1999), "Inhibitory effect of Cordycceps sinensis on spontaneous

liver metastasis of lewis lung carcinoma and B16 melanoma cells in syngeneic

mice ", Jpn. J. Pharmacol, 79, 335-341.

20. R. Guieu, B. Dussol, G. Halimi, G. Bechis, F. Sampieri, Y. Berland, J.

Sampol, F. Couraud and H. Rochat (1998), "Adenosine and the Nervous System:

Pharmacological data and therapeutic perspectives", Gen. Pharmac., Vol. 31, No.

4, 553-561.

21. R. Russell, M. Paterson (2008), "Cordyceps - a traditional Chinese

medicine and another fungal therapeutic biofactory?", Phytochemistry, 69, 1469-

1495.

22. Richard Alan Miller (2009), "The Codyceps sinensis medicinal

mushroom", Nexusmagazine.

23. S.P. Li, F.Q. Yang, Karl W.K. Tsim (2006). "Quality control of Cordyceps

sinensis, a valued traditional Chinese medicine", Journal of Pharmaceutical and

Biomedical Analysis, 41, 1571-1584.

24. The Ministry of Health of the People's Republic of China (2010), The

Pharmacopoeia of the People's Republic of China (PPRC).

25. Xiao Feng Xue, Jin Hui Zhou, Li Ming Wu, Liang Hu Fu, Jing Zhao

(2009), "HPLC determination of adenosine in royal jelly", Food Chemistry,

115(2), 715-719.

PH LC

PH LC A: MT S HNH NH V SC THU C

Ph lc A1: Mt s loi THT

Hnh A1.1. Hnh nh ca Cordyceps capitata

Hnh A1.2. Hnh nh ca Cordyceps ditmarri

Hnh A1.3. Hnh nh ca Cordyceps gracilis v Cordyceps sphecophata

Ph lc A2: Sc kho st khong tuyn tnh v xy dng ng chun

0.0 2.5 5.0 7.5 10.0 12.5 min

0

500

1000

1500

2000

2500mAU

260nm4nm (1.00)

Ad

en

osi

ne

/10

.31

6/1

65

70

46

2

Hnh A2.1. Sc chun 263ppm

0.0 2.5 5.0 7.5 10.0 12.5 min

0

500

1000

1500

2000

mAU260nm4nm (1.00)

Ad

en

osi

ne

/10

.31

1/1

53

79

03

0

Hnh A2.2. Sc chun 210ppm

0.0 2.5 5.0 7.5 10.0 12.5 min

0

250

500

mAU260nm4nm (1.00)

Ad

en

osi

ne

/10

.33

1/3

61

69

64

Hnh A2.3. Sc chun 50,30ppm

0.0 2.5 5.0 7.5 10.0 12.5 min

0

50

100

150

200

mAU260nm4nm (1.00)

Ad

en

osin

e/1

0.3

01

/14

93

08

8


0.0 2.5 5.0 7.5 10.0 12.5 min

0

25

50

75

100

125mAU

260nm4nm (1.00)

Ad

en

osin

e/1

0.3

21

/74

45

75


0.0 2.5 5.0 7.5 10.0 12.5 min

0

25

50

mAU260nm4nm (1.00)

Ad

en

osi

ne

/10

.30

9/3

71

48

9


0.0 2.5 5.0 7.5 10.0 12.5 min-5

0

5

10

15

20

mAU260nm4nm (1.00)

Ad

en

osi

ne

/10

.31

0/1

49

17

0


0.0 2.5 5.0 7.5 10.0 12.5 min

-2.5

0.0

2.5

5.0

7.5

10.0

12.5mAU

260nm4nm (1.00)

Ad

en

osin

e/1

0.3

11

/74

49

5


0.0 2.5 5.0 7.5 10.0 12.5 min

0.0

2.5

5.0

mAU260nm4nm (1.00)

Ad

en

osi

ne

/10

.30

7/3

71

86


0.0 2.5 5.0 7.5 10.0 12.5 min

-1

0

1

2mAU

260nm4nm (1.00)

Ad

en

osi

ne

/10

.30

2/1

55

65


0.0 2.5 5.0 7.5 10.0 12.5 min

-1.0

-0.5

0.0

0.5

mAU260nm4nm (1.00)

Ad

en

osin

e/1

0.3

18

/77

71


Ph lc A3: Sc phn tch mt s mu thc

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

0

100

200

300

400mAU

260nm4nm (1.00)

Ad

en

osi

ne

/10

.17

7/3

45

87

6

Hnh A3.1. Sc mu 08131791nk

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

0

50

100

150

200

250

300

mAU260nm4nm (1.00)

Ad

en

osin

e/1

0.1

75

/77

60

7

Hnh A3.2. Sc mu 11131425dv

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

0

100

200

300

400

mAU260nm4nm (1.00)

Ad

en

osin

e/1

0.1

71

/36

85

38

Hnh A3.3. Sc mu 2116nk

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

0

25

50

75

mAU260nm4nm (1.00)

Ad

en

osi

ne

/10

.17

9/8

43

52

Hnh A3.4. Sc mu 83-2dv

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

0

25

50

75

mAU260nm4nm (1.00)

Ad

en

osi

ne

/10

.20

8/2

03

40

2

Hnh A3.5. Sc mu 776nk

PH LC B: MT S TIU CHUN V CCH NH GI

Ph lc B1: Tiu chun xc nh lp li

i lng o ngha

Gi tr trung bnh

n

ix

xn

i

1

x l gi tr trung bnh s hc ca tp

hp cc gi tr xi, dng c lng xu

hng ca gi tr trung tm (gi tr

thc).

lch chun SD

SD1

)( 2

n

xx tbi

SD ni ln mc dao ng ca cc kt

qu o xi xung quanh gi tr trung bnh,

th hin s sai lch phn tn ca mt

dy kt qu th nghim so vi gi tr

trung bnh ca n.

lch chun tng i RSD (%)

%100% tbx

SDRSD

Biu th s bin ng, phn tn ca cc

kt qu th nghim.

nh gi chm:

Nu theo 1 tiu chun hoc 1 quy trnh chnh thng c cng b RSD (%) th

RSD (%) tnh c phi nh hn hoc bng RSD (%) cho php.

Nu theo 1 tiu chun, 1 quy trnh chnh thng khng cng b th ngi ta

thng so snh RSD (%) tnh c so vi RSD (%) cho php nng cht tng

ng theo AOAC. RSD (%) tnh c khng c ln hn tr gi trong bng hm

lng cht tng ng, chm thay i theo nng cht phn tch. Nng cht

cng thp th kt qu cng dao ng nhiu (khng chm) ngha l RSD cng ln.

Bng B1.1. lp li ti a chp nhn ti cc nng khc nhau (theo AOAC)

TT Hm lng % T l cht n v RSD (%)

1 100 1 100% 1,3

2 10 10-1 10% 1,8

3 1 10-2 1% 2,7

TT Hm lng % T l cht n v RSD (%)

4 0,1 10-3 0,1% 3,7

5 0,01 10-4 100ppm 5,3

6 0,001 10-5 10ppm 7,3

7 0,0001 10-6 1 ppm 11

8 0,00001 10-7 100ppb 15

9 0,000001 10-8 10ppb 21

10 0,0000001 10-9 1ppb 30

Ph lc B2: nh gi thu hi

Thng thng thu hi tnh c phi nm trong khong cho php ca

AOAC nng cht tng ng. Trong trng hp phn tch cc cht hm lng

vt c th tham kho tiu chun ca hi ng chu u.

Bng B2.1. thu hi chp nhn cc nng khc nhau theo AOAC

TT Hm lng (%) T l cht n v thu hi (%)

1 100 1 100% 98-102

2 10 10-1 10% 98-102

3 1 10-2 1% 97-103

4 0,1 10-3 0,1% 95-105

5 0,01 10-4 100ppm 90-107

6 0,001 10-5 10ppm 80-110

7 0,0001 10-6 1 ppm 80-110

8 0,00001 10-7 100ppb 80-110

9 0,000001 10-8 10ppb 60-115

10 0,0000001 10-9 1ppb 40-120

Cn chu u th thu hi cho php theo bng nh sau:

Bng B2.2. Quy nh v thu hi ca hi ng chu u

TT Hm lng cht n v thu hi (%)

1 1 g/kg 1ppb 50-120

2 >1 g/kg n

xác định adenosine trong thực phẩm chức năng có chứa đông trùng hạ thỏa bằng...

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