LIN HIP CC HI KHOA HC V K THUT VIT NAM

TRUNG TM NGHIN CU V PHT TIN CNG NGH HA SINH

BO CO TNG KT

TI:

NGHIN CU S DNG CC HP CHT

POLYPHENOL TRONG MT S GING
CH VIT NAM

8412

H Ni 2010

MC LC
Trang
Tm tt thng tin v ti
Cc k hiu vit tt trong ti
PHN 1: M U
PHN 2: TNG QUAN TI LIU

2.1. MT S C IM CA CC HP CHT POLYPHENOL THC

VT
2.1.1. Flavonoid thc vt
2.1.2. Tanin thc vt
2.2. CY CH VIT NAM
2.2.1. Ngun gc cy ch
2.2.2. Vai tr ca cy ch i vi nn kinh t
2.2.3. Cc vng ch ca Vit Nam
2.2.4. C cu ging ch
2.2.5. Mt s nghin cu trong nc v ngoi nc v ha - sinh y dc
hc trn cy ch (Camellia sinensis O. Kuntz)
PHN 3: I TNG V PHNG PHP NGHIN CU
3.1. I TNG NGHIN CU
3.1.1. i tng thc vt
3.1.2 i tng ng vt
3.2 PHNG PHP NGHIN CU
3.2.1. Chit xut v nh lng Polyphenol tng s theo phng php ca
Folin Denis.
3.2.2. Chit xut v nh lng Flavonoid tng s theo phng php ca B.C.
Talli
3.2.3. nh lng Tanin tng s theo phng php Dc in Vit Nam
3.2.4 Phn tch thnh phn Flavonoid bng sc k lp mng v ph t ngoi
3.2.5 Xc nh hot peroxydaza trong mu theo E. C. Xavron
3.2.6. Nghin cu tc dng khng khun ca cc ch phm Flavonoid chit
xut t cc mu l ch khc nhau
3.2.7. Nghin cu tc dng c t bo invitro ca cc ch phm Flavonoid
chit xut t cc mu l ch khc nhau
3.2.8 Xc nh c tnh cp theo phng php ca A. Wallace Hayes
3.2.9 Nghin cu tc dng bo v gan ca ch phm Flavonoid chit xut t
ch Trung Du l xanh F14 (in vivo)

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10
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24
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27
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3.2.10. Nghin cu tc dng h lipid huyt ca ch phm Flavonoid F14 (chit

xut t ch Trung Du l xanh) trn m hnh tng lipid huyt ni sinh gy bi
Triton WR -1339 trn chut nht trng.
3.2.11. Nghin cu hot tnh chng ung th v chng di cn ung th trn
ng vt thc nghim
3.2.12 X l s liu theo phng php thng k sinh hc
PHN 4: KT QU NGHIN CU
4.1. CC NGHIN CU V HO HC
4.1.1. Kho st s c mt ca nhm cht Polyphenol bng cc phn ng nh
tnh c trng
4.1.2. Xc nh trng lng kh tuyt i ca l ch
4.1.3. nh lng Polyphenol tng s trong l ch
4.1.4. nh lng Tanin tng s trong l ch
4.1.5. Chit xut v nh lng Flavonoid tng s trong l ch
4.1.6. Phn tch thnh phn Flavonoid tng s thu c bng sc k lp mng
v ph t ngoi.
4.2 NGHIN CU TC DNG SINH HC
4.2.1.Nghin cu tc dng chng oxy ho (Antioydant) ca cc ch phm
Flavonoid chit xut t cc ging ch khc nhau i vi nhm mu O ca
ngi.
4.2.2 Nghin cu tc dng khng khun ca ch phm Flavonoid chit xut t
cc ging ch khc nhau.
4.2.3 Nghin cu tc dng gy c t bo trn cc dng t bo ung th ca cc
ch phm Flavonoid chit xut t cc ging ch khc nhau.
4.2.4.Nghin cu c tnh cp ca ch phm Flavonoid chit xut t ch
TDLX
4.2.5. Nghin cu tc dng bo v gan ca ch phm F15
4.2.6. Tc dng h lipid huyt ca F15 trn m hnh tng lipid huyt ni sinh
gy bi Triton WR 1339 trn chut nht trng
4.2.7. Nghin cu nh hng ca ch phm Flavonoid chit xut t ch TDLX
ln s pht trin ung th trn c i chut gy bi dng t bo S -180
PHN 5:KT LUN V KIN NGH
TI LIU THAM KHO

32

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46
46

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Phn 1:M U
Cy ch (Camellia sinensis O. Kuntze) l mt trong nhng loi cy c nh
hng ln n cuc sng nhn loi v c b dy lch s pht trin. Hu nh mi dn
tc trn tri t u bit ung tr; y l loi nc ung ph bin nht mang tnh ton
cu. Vic ung tr truyn thng gip cho con ngi dn dn nhn bit nhng gi tr
ch thc ca ch i vi sc khe; nhng cm nhn ban u ca ngi dng ch lu
nm tng bc kim chng v khng nh thng qua hng trm cng trnh nghin
cu ca cc nh khoa hc trn th gii v cc hp cht thin nhin c trong ch. Cng
vi s pht trin sn xut v ch bin lm ung, mt hng nghin cu khc va
mang tnh truyn thng va mang tnh khoa hc nng gi tr ca cy ch ln tm
cao hn: Cy dc liu vi nhng hp cht thin nhin c nhiu tc dng sinh hc
qu i vi sc khe con ngi. Gi tr dc hc v cht lng cm quan ca ch ph
thuc ch yu vo thnh phn ca n. Cc kt qu nghin cu v thnh phn ha hc
ca ch cho bit c khong 120 130 hot cht khc nhau trong cy ch v chng sp
xp thnh cc nhm sau: nhm ng; nhm pectin; nhm tinh du; protein v axit
amin; cc sc t; cc cht v c; vitamin; cc enzim; cht nha; cc cht hu c; cc
cht thc vt th sinh (polyphenol, tannin, cafein.). Hm lng ca cc cht ny c
th thay i ty theo tng ging ch, iu kin sinh thi, ma v, thi im thu hi
Nhiu cng trnh nghin cu trn th gii v trong nc khng nh:
Polyphenol l mt trong nhng hp cht c ga tr c bit quan trng i vi cht
lng ch ni chung v tc dng sinh dc hc ni ring. Vit Nam vic nghin
cu v ha hc v tc dng sinh dc hc ca cc polyphenol v Flavonoid khai thc
t ch vi mc ch phc v y dc hc l mt hng nghin cu mi; vic khai
thc ng dng l ch xanh cha tr bnh mi dng li kinh nghim dn gian n
gin; cc nghin cu v ch ch yu tp trung lnh vc nng cao nng sut v cht
lng ch, dng ch lm thc phm; cha quan tm khai thc ngun nguyn liu giu
cc cht polyphenol thin nhin t ch vo mc ch phng v cha bnh cho con
ngi.
Vit Nam l mt trong nhng quc gia c din tch trng ch ln, c nhiu vng ch
vi cc c im sinh thi c trng, trng nhiu ging ch khc nhau; v vy vic
nghin cu, iu tra tm ra nhng vng ch, ging ch c hm lng v cht lng
polyphenol u vit nh hng khai thc mt cch c hiu qu ngun nguyn liu
phong ph ny vo mc ch phc v sc khe cng ng l mt hng nghin cu
mi, cn thit v c nhiu trin vng. Vi mong mun ng gp nhng hiu bit
1

nng cao gi tr cy ch Vit Nam v hng ti ng dng cc hp cht Polyphenol t

cy ch Vit Nam vo mc ch bo v sc khe chng ti thc hin ti:
Nghin cu s dng cc hp cht polyphenol trong mt s ging ch trng
Vit Nam

Nhng ni dung chnh ca ti

Chit xut v nh lng cc hp cht polyphenol ch yu trong l ca mt
s ging ch trng Vit Nam
- nh lng Polyphenol tng s
- Chit xut v nh lng Flavonoit tng s ( giu cc cht catechin v 1 t l nh
cc flavonoit khc) v vy gi tn l ch phm giu catechin. Thu 16 ch phm
- nh lng Tanin tng s
- Phn tch cc ch phm giu catechin bng sc k lp mng v ph t ngoi.
Nghin cu tc dng sinh hc ca cc ch phm polyphenol ( tc cc ch
phm giu catechin) chit xut t l ch.
a.Kim tra hot tnh chng oxy ha ca cc ch phm giu catechin thu t ch.
b.Nghin cu hot tnh khng khun ca cc ch phm giu catechin trn cc chng vi
khun kim nh. Chn nhng mu c hot tnh khng khun cao nh lng ( tm
nng c ch ti thiu xc nh MIC, IC50).
c.Nghin cu c tnh t bo ( th trn t bo ung th)
d. T nhng kt qu thu c trn tin hnh sng lc chn ra mt vi mu c hm
lng polyphenol cao, hot tnh sinh hc tt nghin cu tip mt s hot tnh sinh
hc theo nh hng.
- Th c tnh cp, LD50
- Nghin cu tc dng bo v gan trn ng vt thc nghim
- Nghin cu tc dng bo v tim mch ( Th trn ng vt thc nghim v tc dng
iu chnh cholesterol trong mu)
- Nghin cu tc dng chng ung th trn ng vt thc nghim
Tng kt ti
Tng hp s liu v thu thp kt qu t cc chuyn nghin cu. Vit bo co
nghim thu ti.

Phn 2: TNG QUAN TI LIU

2.1. Mt s c im ca cc hp cht polyphenol thc vt [7, 12, 22,23]
Hp cht phenol l cc nhm cht khc nhau rt ph bin trong th gii thc
vt, chng thng tn ti di dng glycozit d tan trong nc v thng tp trung
cc khng bo ca t bo thc vt. c im chung ca chng l trong phn t c
vng thm (vng benzen) mang mt hay hai, ba ...hoc nhiu nhm hydroxyl (OH)
gn trc tip vo vng benzen. Tu thuc vo s lng v v tr tng h ca cc
nhm ny m cc tnh cht l ho hc hoc hot tnh sinh hc thay i.
Da theo s lng nhm hydroxyl m ngi ta phn bit thnh:
*Nhm phenol n gin: gm cc cht c cu to t 1 vng benzen v 1
hay nhiu nhm OH, c phn thnh cc: mono phenol; di phenol (pyrocatechin,
rezoxyn...); tri phenol (pyrogalon, oxy hydroquinon...)
* Nhm hp cht phenol phc tp (polyphenol): trong thnh phn cu to,
ngoi vng benzen cn c d vng mch nhnh, c phn thnh cc nhm: monome
v polyme
+ Monome hay polyphenol n gin: c chia thnh
- Nhm C6 C1 (axit phenol cacbonic): trong cu trc phn t c thm nhm
cacbonyl, thng gp ht ny mm.
-Nhm C6 C3 ( axit cumaric, axit cafeic): c gc cacbonyl c ni vi nhn
benzen qua hai nguyn t cacbon, thng gp thc vt bc cao.
-Nhm C6 C3 C6: gi l cc Flavonoid v c chia thnh cc nhm ph
nh flavon, flavonol (sc t vng), antoxyanidin (sc t xanh, v tm), catechin
(khng mu)...
+Nhm hp cht polyphenol polymer: c chia thnh cc nhm ph nh
Tanin, Lignin, Axit Humic.

Cc cht
phenol

Polyphenol

Phenol n gin

Monophenol

Diphenol

Triphenol

Nhm C6 - C1
Axit phenolcacbonic

Nhm C6 - C3
Axit Cumaric
Axit Cafeic

Tanin

Phenol Polymer

Lignin

Humic

Nhm C6 - C3- C6
Flavonoid
Aglycon: Flavon, Flavonol,
Flavanon, Flavanonol, Chalcon,
Auron, Catechin,
Leucoanthocyanidin, Anthocyanidin,
izoflavonoid, neoflavonoid,
BiFlavonoit

Glycozit:
O - Glycozit
C - Glycozit

S 1: S phn loi cc hp cht Phenol v Polyphenol

Cc hp cht polyphenol chim mt v tr quan trng trong i sng thc vt,
chng tham gia vo nhiu qu trnh sinh l v sinh ho quan trng; vo cc qu trnh
trao i cht di nhiu hnh thc khc nhau nh qu trnh h hp t bo (vn chuyn
H + trong qu trnh photphoryl ho oxy ho ...), qu trnh quang hp, iu ha sinh
trng pht trin ca thc vt.
Thnh phn Polyphenol ca l ch rt a dng, nhng bao gm ch yu l: cc
Flavonoid v tanin. Cc polyphenol ny chim 20 -35 % trng lng ch kh ( l
bp non).
2.1.1. Flavonoid thc vt
Trong s cc polyphenol thin nhin, cc hp cht Flavonoid c ngha thc tin
ln v chng phn b rng ri trong th gii thc vt, t c i vi c th v c nhiu
hot tnh sinh - dc hc gi tr. Cc Flavonoid khng c tng hp ngi v ng
vt; chng c tm thy ng vt l do ng vt n thc vt m c.
Flavonoid c cu to bi khung cacbon CF -C3-C6, gm 2 vng Benzen (vng
A v B) v mt vng pyron (vng C); trong vng A kt hp vi vng C to thnh
khung Chroman.

Trong thc vt Flavonoid tn ti 2 dng: Dng t do (gi l Aglycon) v dng

lin kt vi ng (glycozit). Cc glycozit khi b thy phn bng axit hoc enzym s
gii phng ra ng v aglycon. Ty theo mc oxy ha ca mch 3 cacbon, s c
mt hay khng ca ni i gia C2 - C3 v nhm Cacbonyl C4 m phn loi cc
Aglycon ca Flavonoid thnh cc nhm ph sau:
* Flavon, Flavonol, Flavanon, Flavanonol, Catechin, Leucoanthoxyanidin,
Anthoxyanidin, Chalcon, Auron.
Catechin l hp cht Flavonoid ph bin trong thin nhin v c nhiu trong qu
v l ch.
* Ngoi ra cn c cc dn xut:
- IzoFlavonoid (vng B ni vi vng C v tr C3)
- NeoFlavonoid (vng B ni vi vng C v tr C4)
- BiFlavonoid (2 phn t Flavonoid lin kt vi nhau)
-Cc cng trnh nghin cu cho bit thnh phn Flavonoid trong l ch xanh
ch yu l:
*Catechin v cc dn xut ca catechin nh: catechin, epigallocatechin (EGC),
gallocatechin (GC), epigallocatechin gallat (EGCG), epicatechin (EC),
epicatechin gallat(ECG)
*Cc cht flavonol: Quercetin, Kaempferol, Myricetin v cc dn xut khc ca
flavonol
*Theaflavin v diosmin: Theaflavin sn phm ca s oxy ha ng thi
epigallocatechingalat v epigallocatechin.
-Cn cc Flavonoid trong ch en ch yu l Theaflavin v Thearubigin.
Thearubigin l nhm sn phm oxy ha b polimer ha ca catechin v cc hp cht
gallat ca n.

Cng thc ha hc ca mt s Flavonoid trong l ch xanh (Camellia

sinensis)

HO

2
3

6
5

OH

1'
6'

3'

2'

8
4'

5'

HO

OH

OH

Khung cacbon ca Flavonoid

OH

O
flavonol

R'

kaemferol (R = R' = H)
quercetin (R = OH; R' = H)
myricetin (R = R' = OH)

HO

OH

OH

6
4

OH

OH

catechin

(-) epicatechin

catechingallat

gallocatechingallat

Teaflavin
Teaflavingallat

-Vic nghin cu v cu trc ha hc, cc c im l - ha - sinh hc v kh nng

ng dng ca Flavonoid vo cc lnh vc khc nhau ca i sng tr thnh mt
trng phi ln mnh nhiu quc gia trn th gii (M, Anh, Nga, Hn Quc, Trung
Quc...). Ngi ta tm thy hn 4.000 cht Flavonoid t thc vt (l dn xut ca
cc Aglycon k trn) cng vi tc dng sinh hc ca chng. Tuy nhin, vi mi loi
cy c cc nh nghin cu li lun khm ph ra nhng ci mi v b hp dn bi
nhng tc dng sinh - dc hc ca chng.
Di y l mt s dn liu v tc dng sinh - dc hc tiu biu ca Flavonoid
c cng b trn cc knh thng tin quc t (sch chuyn ngnh, tp ch, hi
ngh...).
a. Tc dng chng oxy ha (antioxydant)

Do bn cht cu to polyphenol nn Flavonoid trong t bo thc vt hoc

trong c th ng vt v ngi chu tc ng ca cc bin i oxy ha - kh, b oxy
ha tng bc v tn ti cc dng hydroquinon, semiquinon, quinon. Nhng
6

Flavonoid c cc nhm hydroxyl sp xp v tr ortho d dng b oxy ha di tc

dng ca cc enzym polyphenoloxydaza v peroxydaza c trong t bo ng, thc vt;
phn ng nh sau:
1. O2 + Flavonoid (kh)

xydaza
polyphenol

Flavonoid (dng b oxy ha) +

H 2O
(Hydroquinon)

H 2O

2. H2O2 + Flavonoid (kh)

(Semiquinon hoc Quinon)

peroxydaza

(Hydroquinon)

Flavonoid (dng b oxy ha) +

(Semiquinon hoc Quinon)

Semiquinon hoc quinon l nhng gc t do bn vng, c th nhn in t v hydro t

nhng cht cho khc nhau tr li dng hydroquinon. Cc cht ny c kh nng phn
ng vi cc gc t do hot ng trit tiu chng. Khi a Flavonoid vo c th s
sinh ra gc t do bn vng hn cc gc t do c hnh thnh trong qu trnh bnh l
(vim nhim, ung th, lo ha...), chng c kh nng gii ta cc in t trn mch
vng ca nhn thm v h thng ni i lin hp, lm trit tiu cc gc t do hot
ng. Cc gc t do to nn bi Flavonoid phn ng vi cc gc t do hot ng v
trung ha chng nn khng tham gia vo dy chuyn phn ng oxy ha tip theo. V
vy Flavonoid c gi l "Nhng tc nhn thu dn v hy dit" cc gc t do c hi.
Tc dng chng oxy ha ca Flavonoid tng dn khi tng nng ca chng v tun
theo trt t fustin < catechin < quercetin < rutin = luteolin < kaempferol < morin.
b.Tc dng i vi enzym
Cc Flavonoid c kh nng tc ng n hot ng ca nhiu h enzym ng
vt trong cc iu kin in vitro v in vivo. Kh nng tng tc vi protein l mt trong
nhng tnh cht quan trng nht ca cc hp cht Polyphenol, quyt nh hot tnh
sinh hc ca chng. Phn ng xy ra gia nhm oxyphenolic (-OH) v oxy cacbonyl
ca cc nhm peptit to thnh lin kt hydro. Tnh bn vng ca lin kt ph thuc
vo s lng v v tr nhm OH v kch thc phn t ca hp cht phenol. Do bn
thn cc cht Flavonoid khi trong c th ng vt tn ti dng oxy ha hoc kh
v chu nhiu bin i phc tp cho nn c th trong cc iu kin khc nhau n s th
hin hot tnh sinh hc khc nhau: km hm hoc kch thch hot ng enzym, hoc
kch thch c mc v c iu kin - theo nhng c ch phc tp hn trong nhng
nghin cu in vitro.
c. Tc dng khng sinh, chng vim nhim
Tc dng chng vim nhim v khng khun ca Flavonoid c nhiu cng
trnh nghin cu trn th gii chng minh. Mt s tc gi nghin cu tc dng ca 24
anthoxyanin, leucoanthoxyanin v axit phenolic ln Salmonella (vi khun thng hn)
7

v thy c tc dng km hm r rt. Hu ht cc cht ny u c kh nng km hm s

h hp hay phn chia ca vi khun khi c mt glucoza. Tc dng chng giun ca
Flavonoid c thng k vi 16 chalcon v nhng cht tng t, c bit l cc
chalcon c nhiu nhm th OH. Tc dng khng virut ca Flavonoid cng c
khng nh; Chang-Qi Hu v cng s nghin cu kh nng c ch virut HIV cc
t bo H9 ca 35 Flavonoid chit xut t thc vt v tng hp - thy rng cc hp
cht c ni i v tr C2 = C3 v c nhm OH C6 v C4 th hin hot tnh cao hn
cc Orthodiphenol. Trong cc dng t bo ngi, Herpesvirus hominis b km hm bi
quercetin nng 300g/ml, nhng khng b km hm bi rutin hay
dihydroquercetin. Khi thm quercetin vo virut gy bnh ngi, virut s b km hm
nu n c v bc v cht nu khng c v bao bc bo v.
d. Tc dng i vi cc bnh tim mch
Tc ng bo v ca cc Flavonoid i vi tim mch c th do kh nng ca
chng trong vic ngn nga s oxy ha cc lipoprotein t trng thp, phng nga x
va ng mch, chn s kt t huyt khi, iu ha nhp tim, ngn nga bnh mch
vnh v nhi mu c tim, iu ha huyt p...
e. Tc dng i vi ung th [ 17,18]
Ti Vin L Ha Vin Hn lm khoa hc Lin X (c) hnh thnh nhng
phng hng mi trong lnh vc tm kim thuc chng ung th da trn quan nim
v vai tr ca cc gc t do trong c ch bin i nhng t bo bnh thng thnh t
bo ung th v s pht trin tip theo ca qu trnh ung th. Cc tc gi s dng
nhng hp cht flavonoid hoc polyphenol c c tnh thp nh nhng cht chng oxy
ha nghin cu lm sng iu tr mt s dng ung th. Ch phm ionol v gosipol
(bn cht flavonoid t mt loi cy bng) c ng dng iu tr ung th bng
quang v em li kt qu kh quan (vi 6 trong 54 bnh nhn mt hon ton khi u, 6
bnh nhn khc thy khi u nh kch thc mt cch ng k). Ngi ta cho rng c
ch chng khi u ca flavonoid khng ch do kh nng chng oxy ha m cn tc
dng tng hp do kh nng phn ng a dng ca phn t flavonoid.
Cc cng trnh nghin cu ca Racker v cng s cho bit cc t bo c tnh
gy bi virut (kiu Rous sarcoma virut) c lin quan n tc dng ca cc kinaza. Khi
cc t bo ung th nui cy vi flavonoid, cc t bo c tnh tr li thnh cc t bo
bnh thng do flavonoid lm thay i hot tnh ca cc kinaza.
Vi n lc nhm nghin cu kh nng chng ung th ca cc hp cht
flavonoid, gn y nhiu tc gi trn th gii tip tc th nghim tc dng invitro
v invivo ca flavonoid ln cc dng t bo ung th khc nhau, Byung- Zun v GynYong Song [36] nghin cu tc dng ca 46 flavon tng hp ln cc dng t bo
8

ung th khc nhau v rt ra nhn xt cc hp cht 2-benzyloxy-5 metoxyflavon, 2benzyloxy- 5,7-dimetoxyflavon, 2-benzyloxy 5,7,8 trimetoxyflavon, 2-benzyloxy
5-hydroxyflavon,5,2-dihydroxyflavon th hin tnh gy c t bo i vi dng t
bo LI210 v HL-60. Tc dng chng khi u gy bi dng t bo ung th S-180 trn
chut i vi 52-dihydoxy 6,7,8,6- tetrametoxyflavon chit xut t
Scutellariabaicallensis dc chng minh bi B.Ahn v cng s. Bng cc chuyn
ha cu trc vng B, mt s tc gi nhn thy c mi lin quan cht ch gia hot tnh
chng khi u ca hp cht flavonoid trn vi s c mt ca nhm th oxy v tr 2
hay hai nhm th oxy v tr 2,6 ca vng B v s i gc lin kt gia vng B v C.
g. Tc dng i vi chuyn ha v trn lm sng

Cc Flavonoid c hp th theo ng d dy - rut ngi v ng vt, v

c bi tit nguyn dng hoc di dng cht chuyn ha ca chng qua nc tiu v
phn. Mt vi Flavonoid dng glycozit b deglycosyl ha bi cc enzym trong m ca
ngi. Tc v phm vi deglycosyl ha ph thuc vo cu trc Flavonoid v v tr,
bn cht ca gc ng b thay th bng php o kh nng chng oxy ha ca huyt
tng v nc tiu sau khi chuyn ha. Ch xanh cho thy s hp th cc cht oxy ha
trong Ch xanh din ra nhanh. Cc cht chng oxy ha i vo vng tun hon ngay sau
khi ung vo lm cho lng cht chng oxy ha trong huyt tng tng ln ng k.
Benzie v cs (1999) cho thy rng s tng hm lng Flavonoid ch xanh trong
huyt tng c th lm gim s thit hi do cc phn ng oxy ha gy ra v v th lm
gim nguy c mc ung th.
Cc Flavonoid c tc ng su sc ln chc nng ca cc t bo min dch v t
bo vim. Trong cc nghin cu trn ng vt hai este methyl EGCG tch chit t Ch
long gy km hm ng k ln cc phn ng d ng trn chut. Gaby (1998) ch ra
rng quercetin c th c gi tr trong iu tr hen suyn v tt cho cc bnh nhn tiu
ng v nhim HIV (human immunodeficiency virus). Flavonoid baicalin trong cc
nghin cu gn y cho thy c hot tnh chng vim v chng HIV - 1 theo c ch
gy cn tr s tng tc gia cc protein v ca HIV -1 vi cc receptor v ngn chn
HIV -1 tn cng vo cc t bo ch.
h. Mt s ng dng ca Flavonoid trong y hc: nhng Flavonoid c hot tnh sinh
hc c gi l Bioflavonoid. Trong y hc lm sng, tc dng ca Bioflavonoid rt a
dng, di y l mt s phng hng ng dng chnh:
-Bo v thnh mch, phng chng nguy c chy mu.
-Chng d ng.
-Chng vim lot, khng khun, khng nm.
-iu tr nhiu bnh v gan, mt
-Phng v cha mt s bnh lin quan n chuyn ho
9

-iu ha hm lng cc dng cholesterol trong mu, trnh nguy c x va

mch; phc hi trng lc c tim, iu ho nhp tim v huyt p; iu ho chuyn ha
canxi
-Mt s bnh lin quan n ni tit v iu ho cn bng sinh hc ca c th.
-H tr cho c th khi dng nhiu khng sinh hoc sng trong mi trng c
nhiu bc x ion.
-V phng din min dch hc, nhiu bioFlavonoid c kh nng kch thch
limpo t bo sn xut inteferon, chng virus xm nhp vo c th v km hm s nhn
ln ca virus.
-Gim au do tc dng chng co tht, gin c trn. Lm gim cc m xut
huyt nh trong bnh i ng.
- Mt s Flavonoid c tc dng chng khi u lnh v c tnh. Lm tng to mu
do lm tng tng hp axit folic ca vi khun ng rut, tng s lng hng cu v t
l hemoglobin.
-Loi tr ri lon thn kinh c do thiu vitamin C.
2.1.2. Tanin thc vt
-Tanin (hay cht cht) rt ph bin trong c th thc vt, cc tanin tuy c cu
trc ha hc khc nhau nhng u c bn cht chung l polyphenol. Tanin thin nhin
u l hp cht ca axit gallic v digallic dng t do cng nh dng kt hp vi
glucoza. Di tc dng ca tanin, protein s ng vn, da cn nguyn s thnh da
thuc ngha l to thnh da rt bn vi nc v vi vi sinh vt gy thi, c tnh do,
tnh n hi
-Ngi ta thng chia Tanin thnh 2 nhm l: Tanin thy phn pyrogalic
(Tanin Gallic) v Tanin ngng t (Tanin Catechin).
a. Tanin Gallic (Tanin thy phn pyrogalic)

-Thuc nhm ny c cc tanin m thnh phn chnh to polymer thng l

este ca axit gallic vi gc ng, cc este khng mang ng ca axit
phenolcacbonic v cc este ca axit ellagovic vi ng.
* Este ca axit gallic vi gc ng
- Thng gi l gallotanin v n l nhm quan trng nht trong cc tanin thy
phn, ngi ta thng gp n dng di, tri, tetra, penta hoc dng polygallic este
(nhiu phn t axit gallic lin kt vi gc ng to thnh Tanin)
-i din ca nhm ny l - pentadigalloyl D glucoza chit t mt cy ht
trn Trung Quc c nhiu ng dng trong i sng nn hay c gi tt l Tanin
Trung quc.
* Este ca axit phenolcacbonic
-Cc tanin ca nhm ny khng to este vi cc gc ng m thay vo l
axit gallic to este vi axit phenolcacbonic.
10

- i din ca nhm ny l Teogallin l mt polyoxiphenol v c trong ch

ti

Teogallin
* Este ca axit ellagovic vi ng
-Cc Tanin thuc nhm ny thng phc tp hn v c nhiu thc vt nhit
i. N l este ca axit ellagovic vi ng, cc axit gallic l sn phm u tin to
ra axit ellagovic.
b. Tanin Catechin (Tanin ngng t)

- L hp cht do cc catechin ngng t vi nhau theo kiu u ni ui (C6

C8 vi catechin) hoc kiu ui ni ui (C6 C6, vi galocatechin) hoc kiu
u ni u ( C4 C8, vi flavandiol -3,4) to thnh. Tanin ngng t khng b phn
gii bi axit v c cng nh bi enzim.
- i din ca nhm ny l tanin c trong ch: l hn hp polymer ca catechin
v leucoanthoxyanidin, c cng thc nh sau

11

Cht lng cm quan ca ch ph thuc kh nhiu vo hp cht tanin, bi n chnh l

hp cht to ra v cht c trng khng th thiu ca cy ch. cht cao hay thp ty
thuc vo t l tanin v cc cht ha tan c trong ch. C th ni khng sai lm l tt
c nhng thuc tnh c bn ca nc ch nh: mu, mi, v u lin quan t nhiu n
tanin v cc dn xut ca tanin. Tanin trong cy ch ch yu l tanin catechin nn
ngoi c tnh qui nh cht lng cm quan n cn l hp cht c nhiu hot tnh sinh
dc hc.
c. Tc dng sinh hc ca Tanin

- Tanin lm cho ming c cm gic kh, se, lm cho biu b cng li v gim s bi
tit. Cho nn tanin thng dng ch bin thuc sc ming, thuc tht m h hay
niu o.
-Mc d tanin c tnh gy dn mch, nhng li c tc dng nh mt cht cm mu nh,
dng trong s c chy mu mao qun (n kt hp vi protein to thnh cc mu)
- Do tanin c tnh st trng nn n cn tr cc qu trnh ln men ca vi sinh vt, n c
kh nng bnh thng ha hot ng ca h vi khun c ch trong rut, ngn nga qu
trnh thi ra, sinh hi v nhng ri lon khc lm cn tr hot ng ca rut.
-Tanin c tnh cht gy kt ta vi ancaloit nn n cn c dng trong cc trng
hp ng c do ancaloit.

2.2. Cy ch Vit Nam [ 1,3,4,5,12,13]

Cy ch Vit Nam c tn khoa hc l Camellia sinensis (L) O. Kuntze (tn
ng ngha l Thea sinensis L) thuc nghnh ht trn Angiospermae, lp 2 l mm
Dicotyledenae, b ch Theales, h ch Theaceae, chi ch Thea (ng ngha vi
Camellia), loi Camellia (Thea) sinensis.
Cy ch sinh trng tt trong cc iu kin kh hu, t ai sau y:
- Nhit bnh qun nm: 15 250 C
- Lng ma bnh qun nm: 1500 2000 mm
- m tng i ca khng kh: 80 85%
- t c phn ng chua pH 4,5 6, tng dy 1m, nhiu mn v giu cht dinh
dng N, P, K, nht l Nit. Kt cu t ti xp, dc thoi, va thong va gi c
nc, thuc loi t tht.
2.2.1. Ngun gc cy ch
T xa xa, ngi ta cho rng ngun gc ca cy ch l vng cao nguyn Vn
Nam Trung Quc, ni c kh hu m t v m. Theo nhiu ti liu th cch y
khong 4000 nm, ngi Trung Quc bit dng ch lm dc liu v sau
mi dng ung, v vng bin gii Ty Bc nc ta nm trong vng nguyn sn ca
ging ch t nhin trn th gii. Tuy nhin, nhng cng trnh nghin cu ca
jemukhatze (1961 1976) v hp cht catechin (thnh phn Polyphenol c trng
12

ca cy ch) c trong l ch t cc ngun gc khc nhau - so snh v thnh phn cc

cht catechin gia cc loi ch c trng v ch mc hoang di nu ln lun im
v s tin ha sinh ha ca cy ch v trn quan im xc minh ngun gc cy ch.
jemukhatze kt lun rng: nhng cy ch mc hoang di t c xa, tng hp ch
yu l (-) epicatechin v (-) epicatechin gallat, chng pht trin chm kh
nng tng hp (-)epigallo catechin v cc gallat ca n to thnh e (+)gallocatechin.
Khi nghin cu v cc cy ch di Vit Nam (rng ch di Sui Ging, Tin
Thng v Thng Nguyn) cho thy chng cng tng hp ch yu l: (-) epicatechin
v (-) epicatechin gallat (chim 70% tng s cc loi catechin). Khi di thc cy ch
di ny ln pha Bc, vi cc iu kin khc nghit hn v kh hu, chng s thch ng
dn vi cc iu kin sinh thi bng cch c thnh phn catechin phc tp hn, cng
vi s to thnh (-)epigallocatechin v cc gallat ca chng. iu ny c ngha l s
trao i cht y hng v pha tng cng qu trnh hydroxyl ha e (-)
epicatechin v gallic ha. Theo cc kt qu nghin cu , th cy ch c Vit Nam
tng hp cc cht catechin n gin nhiu hn cy ch Vn Nam Trung Quc v s
tin ha ca cy ch th gii l nh sau: Camellia ch Vit Nam ch Vn Nam l
to ch Assam (n ).
Ngy nay, cy ch c phn b kh rng ri trong nhng iu kin t nhin
rt khc nhau t 30 v nam (Natan Nam phi ) n 45 v bc (Gruria Lin X
c) l nhng ni c iu kin t nhin khc rt xa vng nguyn sn. Hin ti c
khong 40 nc trng ch, ch c tp trung trng nhiu nht chu sau n
chu Phi. Nhng nc trng ch ph bin l: Trung Quc, n , Srilanca, Nht Bn,
Indnxia, Nga, i Loan, Vit Nam.
2.2.2. Vai tr ca cy ch i vi nn kinh t
Ch l mt cy cng nghip lu nm, c i sng kinh t lu di, nhanh cho sn
phm, hiu qu kinh t cao. Ch trng mt ln c th thu hoch 30 40 nm hoc lu
hn na. Trong iu kin thun li cy sinh trng tt th cui nm th nht thu bi
trn di 1 tn bp ti / ha. Cc nm th 2 th 3 (trong thi k kin thit c bn)
cng cho mt sn lng ng k, khong 2- 3 tn bp ti / ha. T nm th 4 ch
a vo kinh doanh sn xut. Hn na ch li l sn phm c th trng quc t n
nh, rng ln v ngy cng c m rng.
Vit Nam l mt trong nhng nc c tim nng v iu kin thun li pht
trin sn xut ch: din tch t theo qui hoch trng ch rt ln (trn 200.000
ha); iu kin kh hu rt ph hp cho cy ch pht trin: lm nng, ma nhiu lng nc ma trung bnh 1700 2000mm/ nm, m khng kh 80 85%,
nhit trung bnh 21 22,60C; t trng ch gm hai loi l phin thch st v
bazan mu m y chnh l nhng iu kin thun li cho s sinh trng v pht
13

trin ca cy ch v l iu kin tt cy ch tng hp c cc hot cht c trng,

to ra nt khc bit v cht lng cm quan v tc dng dc hc ca ch Vit Nam.
Hn na, nhn dn ta c tp qun trng v ung ch t lu i, c rt ra nhiu
kinh nghim sn xut v ch bin ch. Mt khc nh nc cng rt coi trng vic pht
trin ca ngnh ch, coi ch l mt nghnh kinh t mi nhn ca trung du v min ni.
Theo thng k ca Hip hi ch Vit Nam, tnh n u nm 2008 Vit Nam c din
tch trng ch l 131.090 ha - ng hng th 7 trn th gii, sn lng ch kh ng
th 11 v xut khu ng hng th 5 trong s 30 nc sn xut ch trn th gii.
Trong 8 thng u nm 2008, Vit Nam xut khu c 62, 073 tn ch cc loi t
kim nghch 84, 6 triu USD. Trong ch xanh chim 35,8%, cn li l ch cc loi.
Th trng xut khu ca ch Vit Nam ch yu l cc nc: i Loan, M, Anh, n
, Nga, Trung Quc
2.2.3. Cc vng ch ca Vit Nam
- Vit Nam, cy ch c kh nng thch nghi rng t cc tnh Lm ng n H
Giang, nhng c trng tp trung ch yu su vng ch:
(1) Vng ch Ty Bc (min ni pha Bc): gm 2 tnh l Lai Chu, Sn La
l vng ch ni ting vi cc ging ch Shan.
(2) Vng ch Vit Bc - Hong Lin Sn: gm cc tnh H Giang, Tuyn
Quang, Lo Cai, pha ty Yn Bi (Ngha L, Vn Chn) l vng ch rt ni ting
vi ging ch Shan Tuyt.
(3) Vng ch Trung Du Bc B: gm cc tnh Vnh Phc, Ph Th, Bc Cn,
Thi Nguyn, H Ty, Ha Bnh, Bc Giang, Bc Ninh. Vng ny c loi ch to
c thng hiu ni ting trn c th trng trong nc v quc t l ch Tn Cng
Thi Nguyn
(4) Vng ch Bc Trung B: gm cc tnh Thanh Ha, Ngh An, H Tnh.
(5) Vng ch Ty Nguyn: gm cc tnh Gia Lai Kontum, c Lc, Lm
ng.
(6) Vng ch Duyn Hi min Trung: gm cc tnh Qung Bnh, qung Tr,
Tha Thin, Qung Nam, Qung Ngi, Bnh nh.
- Cn nu tnh theo cao ngi ta thng chia thnh thnh 3 vng sau: vng thp <
300 m, vng gia 300 600 m, vng cao t 600 trn 1000 m
- Tuy nhin, Vit Nam ngi ta thng ni nhiu ti 3 vng ch c sn ni ting
trong c nc v quc t l: vng ch Thi Nguyn (Tn Cng), vng ch Shan
(Mc Chu, H Giang, Lo Cai, Yn Bi) v vng ch Bo LoLm ng
* Vng ch Thi Nguyn

14

- L vng c sn lng ch ln th 2 trong c nc v rt ni ting vi sn

phm ch c sn Tn Cng. Thi Nguyn hin c 16746 ha ch, trng ch yu l
cc ging Trung du ( TDLX, LDP1)
*Vng ch Lm ng
-Vng Ty Nguyn c kh hu nhit i gi ma, b nh hng ln bi dy
Trng Sn chn, m khng kh thp (70%), bin gia ngy v m cao, nhit
trung bnh l 15 -16, t Ty Nguyn l loi t Feralic pht trin trn Bazan.
Vi iu kin kh hu v th nhng nh trn tnh Ty Nguyn c bit l Lm ng
l ni l tng cho cy ch pht trin v c cht lng tuyt ho.
-Tng din tch trng ch ca Lm ng l 23.940 ha (theo s liu ngy
31/12/2002), gm c cc vng nh Bo Lc, Di Linh, Lm H, Cu t, Bo Lm.
Vng ch ny rt c trng l trng c cc ging ch sn xut ch Olong.( 3
ging ch i loan.)
*Vng ch Shan Tuyt (Mc Chu, H Giang, Yn Bi, Bc Cn):
- H Giang l ci ni ca cy ch Vit Nam v cng l ni ca ch th gii, ch
y mi c nhng cy ch Shan c th c tui th hng trm nm, c nhng cy ch
thn to ti mc hai ngi m khng xu. Din tch trng ch ca H Giang l 14.388
ha, trng tp trung cc x, cc huyn vng cao ca tnh nh: Hong Su Ph, X Mn
v cc x ca huyn Bc Quang...Cy ch Shan y c trng cao >1500 m,
chu c lnh nn to ra sn phm ch c cht lng rt c bit; Ngoi ch Shan
tuyt nng trng ch Mc chu cn trng nhiu ging ch nh Shan Nm ngt, Shan
cht tin
Cy ch chu nh hng rt ln do tc ng ca cc iu kin sinh thi trong
qu trnh sng ca n. cc vng sinh thi khc nhau, cy ch chu s tc ng khc
nhau ca cc yu t sinh thi nh nhit , nh sng, m, lng ma . do dn
ti s tng hp v tch ly cc hp cht t nhin ca cy ch cc vng sinh thi khc
nhau l khc nhau, v y chnh l nguyn nhn to ra tnh ring bit v cht lng
cm quan cng nh hot tnh sinh dc hc ca cc hot cht trong ch tng vng
a l khc nhau.
2.2.4. C cu ging ch
V c cu ging ch, hin nay Qu gen ca Tp on ging ch c khong
150 ging ch, trong c ch yu l: cc ging ch bn a nh nhm ging ch
Trung du, nhm ging ch Shan, PH1, LDP1, LDP2, A1, ; cc ging ch nhp ni
ca i Loan, Trung Quc nh ch Kim tuyn, ch Thy ngc, T qu, Olong
Thanh tm, Hng nh bch tr, Thit bo tr, Bt tin, i bch tr, Vn xng, Keo
am tch ;cc ging ch nhp ca Nht Bn nh Yabukita, Menori; cc ging ch
nhp ca n nh Manipur, Assami, PT 95 ; cc ging ch nhp ca
15

Indonexia nh TC1, TC2, TC3, TC4, TC5; cc ging ch nhp ca Srilanca nh

Cynyrual 143, .
Di y l mt s ging ch hin ang c trng ph bin cc vng ch
ca Vit Nam.
a.Nhm ging ch Trung du
- L cc ging a phng hn hp, lai to, khng ng u, nhiu bin d, gm
c cc loi l Trung du l vng, Trung du l xanh, Trung du l xanh thm.
- y l nhm ging ch c trng kh ph bin vng Trung Du Bc B.
Hin ti din tch trng khong 63.000 ha chim 48 % tng din tch trng ch ca
nc ta (Tng din tch trng ch ca nc ta tnh n 2008 l 131.090 ha)
- Dng xn xut ch xanh v ch en
- c im: thn g nh, l to trung bnh, chiu di 12 -14 cm, chiu rng c th
ti 5-7 cm, c10 i gn l ni r lm b mt l gn sng, ging ch ny c sc sinh
trng mnh nhng km chu bnh.
- Nng xut trung bnh: ch 10 -25 nm tui c thm canh tt cho nng sut 5
6 tn bp ti / ha, ch t xu km thm canh ch t nng sut 2, 5 3 tn bp
ti / ha. Tuy nhin, s bin ng nng sut bnh qun rt ln ty thuc vo tng iu
kin kh hu, t ai, ch chm sc ca tng nng ch, ca tng vng ch c th.
b. Nhm ging ch Shan
- L ging a phng, thuc th ch Shan, ging hn hp lai tp, rt nhiu
bin d, c ngi dn a phng chia thnh cc loi: ch Shan Tuyt trng (bp c
lng trng nh tuyt), ch Shan l xanh, ch Shan l vng, hoc ty theo tng a danh
m c cc tn gi khc nhau nh: Shan Tham V, Shan Trn Ninh, Shan Hong Su Ph,
Shan Bc H, Shan Bc Quang, Shan Mc Chu, Shan Sui Ging, Shan Nm Ngt,
Shan Cht Tin
- c trng vi din tch l khong 35.000 ha, chim khong 26,7% tng din
tch trng ch ca Vit Nam. Trng ph bin cc vng ni cao ca H Giang, Sui
Ging, Mc Chu
- Dng sn xut ch xanh v ch en v c th trng quc t c bit a
chung.
- c im: l c mu xanh nht, to, chiu rng 47 cm, chiu di 1617 cm,
c 13 15 i gn l. Chiu cao cy t 8 10 m hoc cao hn. nhng kh nng ra
hoa kt qu yu, chu c lnh (-5- 6 C). L ging c nng xut cao, c th t
c 19 tn bp ti /ha
c. Ging ch LDP1 v LDP2
- L 2 ging ch lai. M l ging ch Trung Quc i Bch Tr - ging c cht
lng thm, ngon ni ting ca Trung Quc nhng nng sut thp (2- 3 tn bp ti /
16

ha). B l ging PH1(ch ph h 1) v PH2 (ch ph h 2) l cc ging c nng sut

cao (t trn 10 tn bp ti / ha), nhng cht lng trung bnh. C tnh thch ng
rng t vng c kh hu m nng n vng lnh.
- Hin ti c trng vi din tch l khong 18.000 ha, chim khong 13,7%
tng din tch trng ch ca Vit Nam
- c im: l ch hnh bu dc n bu dc di, dy, mu xanh nht. Bp ra sm
v kt thc mun, mi t bp sinh trng ngn hn 2- 6 ngy so vi b m. Bp ch
LDP1 v LDP2 thuc loi hnh nh n trung bnh.
- Dng sn xut ch xanh
- Nng sut: ch 4 tui t 4 tn bp ti / ha, ch 5 tui t 6, 7 tn bp ti / ha,
ch 8 tui tr nn t 9 10 tn bp ti / ha.
d. Ging ch PH1
- L ging c to ra bng phng php chn lc c th t tp on Assamica
(n ). Din tch trng hin nay l khong 11.000 ha, chim 8,3% tng din tch
trng ch ca Vit Nam. Trng ph bin vng i ni thp bc b, khu bn c, cao
nguyn trung b (Bo Lc Lm ng).
- C c im: phn cnh thp, s cnh cp 1 nhiu, l mu xanh m hnh bu
dc, l to trung bnh (35 - 40 cm2). Trng lng bp 0, 8 1 g. Tn rng 1,0 -1,4 m.
- Nng xut cao nhng cht lng khng tt lm. Nng xut trung bnh l 15
17 tn bp ti / ha, thm canh tt c th t 25 28 tn bp ti / ha
- Dng sn xut ch en l ch yu
e. Ging ch Kim Tuyn, Bt Tin, Thanh Tm
-L ging ch nhp ni ca i Loan.
-Dng sn xut ch Olong, hin ang c pht trin trng m rng cc
tnh Lm ng, Mc Chu. y l nhng ging ch rt c li th trong vic xut khu
ch i Chu u v cc nc ng Nam . Din tch trng khong > 2000 ha.
h. Cc ging ch khc
- Bao gm nhiu loi ging, tnh n hin nay din tch trng khong 1890 ha,
chim 1,7% tng din tch trng ch ca Vit Nam
- Trong c cc ging c trng kh ph bin nh: TH 3, 1A, TRI 777, v
cc ging c quan tm c bit l: Phc Vn Tin, Keo Am Tch, Hng nh Bch
Tr, Cynyrual 143.
2.2.5. Mt s nghin cu trong nc v ngoi nc v ha - sinh y dc hc trn
cy ch (Camellia sinensis O. Kuntz)
a.Cc nghin cu ngoi nc
Ngoi nhng nghin cu lin quan n vn trng trt v ch bin ch, c
hng nghn cng trnh nghin cu ca cc tc gi nhiu nc trn th gii cng b
17

tc dng cha bnh ca ch xanh. Trong cy ch ngi ta quan tm ch yu n cc

hp cht polyphenol (bao gm Flavonoid catechin v cc Flavonoid khc v tanin)
bi chng c nhiu tc dng sinh dc hc ng qu nh: tc dng chng ung th,
chng t bin t bo, chng phng x, chng vim ... Cc polyphenol ny chim 20
30 % trng lng ch kh. Ch l ngun cung cp cc hp cht Flavonoid chnh cho
con ngi bi ch c s dng nhiu v cha hm lng Flavonoid tng i cao.
Cc Flavonoid tm thy trong ch xanh ch yu l cc flavan 3- ols (catechin), chim
90 % trong cc hp cht phenol ca l ch. Cc Flavonoid trong ch en ch yu l
theaflavin v thearubigin phc hp cc sn phm oxy ha ca cc hp cht phenol
ch xanh. Thearubigin l nhm sn phm oxy ha b polimer ha ca catechin v
cc hp cht galat [18].
Cc Flavonoid c coi l cc cht chng oxy ha mc cao bi chng c
kh nng loi b cc gc t do v cc gc oxy hot ng. Tuy nhin, nhiu nghin
cu cho thy rng hot tnh chng oxy ha ca cc Flavonoid khc nhau l khc
nhau. Quercetin v 5,3,4 trihydroxyl 7- methoxyflavon th hin hiu ng ng
vn khi chng c th dng hn hp. Salah v cng s (1995) ch ra rng hot
tnh chng oxy ha ton phn v th t hiu qu ca cc polyphenol ch xanh trong
vic loi b cc gc t do hot ng c hi l: ECG > EGCG > EGC > axit Galic >
EC = catechin. S oxy ha cc lipoprotein phn t thp b km hm bi catechin, EC,
ECG v EGCG vi mc tng ng, nhng khng bng khi c mt EGC hoc
axit Gallic. Theo nghin mi nht ca cc nh khoa hc trung tm dinh dng
Unilever (Vlaardingen, H Lan), trong nc ch c cha mt lng ln cc cht chng
oxy ha nh catechin (trong ch xanh) v thearubigin (trong ch en) v chnh l
nhng cht chng oxy ha hu hiu gip con ngi chng li nhiu loi bnh tt, c
bit l bnh tim mch. Tin s Rianne Leenen ch nhim trng trnh nghin cu
cho bit, nhng ngi tham gia qu trnh th nghim u c la chn rt k p
ng y cc nguyn tc: i ng tnh nguyn vin ny phi dn u trong
tui t 18 n 70, phi khe mnh v khng ht thuc l; cc kt qu thu c cho
thy: sau khi ung u n mi ngy 3 chn nc ch, c hoc khng c sa ty
thuc vo khu v ca tng ngi th nhn thy s lng v phm vi hot ng ca
cc cht chng oxy ha trong huyt thanh ca c th h tng ln r rt so vi
nhm chng l nhng ngi khng ung nc ch. ng thi nhm nghin cu cn
nhn thy hot tnh chng oxy ha ca ch xanh mnh nht (mnh gp 5 ln ch en),
tip n l ch Olong v ch en th hin hot tnh thp nht.
Hara ch ra rng thi quen ung nc ch c th ngn nga cc bnh tim
mch thng qua vic lm tng kh nng chng oxy ha huyt tng ngi. Hp dn
hn l cc polyphenol ch c hp th nhanh sau khi ung vi sa v sa khng lm
18

gim cc c tnh sinh hc ca polyphenol. Thi quen a vo c th cc Flavonoid t

cc ngun thc ung nh ch cng c th bo v c th khi s t qu hoc lm gim
thp cc nguy c do cc bnh s va ng mch v bnh mch vnh. Ch xanh v ch
en c th bo v c th chng li c tnh oxyt nitric v th c lin quan gia tc ng
c li ca vic ung ch vi s ngn nga cc bnh mch vnh. Thm vo , dng
quercetin c th bo v c th khi cc bnh tim mch bng cch iu tr tnh d v
mao mch v c ch s kt dnh tiu cu. Tc dng khng khun, chng vim lot,
chng t bin ca polyphenol ch i vi nhiu loi t bin c chng minh
trn cc h vi khun, h thng t bo ng vt c v v qua cc test th in vivo ng
vt 21, 30]. Tanin trong ch c tc dng khng khun rt tt, n c kh nng bnh
thng ha hot ng ca h vi khun c ch trong rut, ngn nga qu trnh thi ra,
snh hi v cc ri lon khc lm cn tr hot ng ca rut. Cc polyphenol ca ch
c kh nng c ch nhiu loi vi khun gy bnh nh: clostridium, escherichia,
plassiomonas, pseudomonas, salmonella Thng 8/ 1996 gio s T. Shimamura
(Nht Bn) c cng trnh din thuyt v tc ng dit khun Ecoli 157 hi
tho chuyn dit khun ca tr xanh. Theo ng, catechin ca ch xanh chnh
l hp cht c tc dng khng khun rt tt.
Nhng nm gn y, thng c nhiu cng trnh nghin cu tp trung vo tc
dng ngn nga ung th ca cc cht chng oxy ha t nhin c trong thc n v rau
qu. Nhiu nghin cu chng minh rng ch xanh vi thnh phn ch yu l cc
polyphenol v c bit l EGCG (Epigallocatechingallat) c kh nng c ch s pht
trin ca mt s dng t bo ung th ngi, bao gm t bo ung th ca phi, ph
qun, tuyn tin lit, tuyn v, gan v bch cu. Mt s nghin cu dch t hc
Nht mt trong nhng quc gia s dng ch nhiu nht, ch ra mi quan h t l
nghch gia thi quen ung ch xanh v t l ngi cht v ung th. Qua cho thy
nhm ngi c thi quen ung ch xanh c t l cht do ung th c tnh thp mt cch
ng ngc nhin. Cc nghin cu cng a ra mi lin quan gia mc tiu th ch v
t l mc bnh ung th. Mt s cng trnh nghin cu khc chng minh tc dng
km hm ca polyphenol ch ln s hnh thnh, pht trin v di cn ca khi u. Tc
dng ny ch yu l do hiu qu chng oxy ha cao v chng tng sinh khi u ca cc
hp cht polyphenol trong ch. Ch xanh c th bo v c th khi ung th bng cch
lm ngng chu trnh t bo, k c chu trnh nguyn phn ca t bo ung th. Nhiu
nghin cu cp n 2 c ch chnh v tc dng c ch s pht trin t bo u ca
EGCG: (1) tc dng ngn cn chu k phn chia t bo, dng chu k phn chia t
bo cc pha G1, G2; (2) thc y qu trnh apoptosis (s cht t bo theo chng
trnh). Cc nghin cu trn m bnh hc cho thy rng c ch xanh v ch en u c
th km hm s tng sinh ca cc t bo u trong cc m hnh ng vt. EGCG, EGC v
19

ECG km hm lipoxygenaza u tng mt nhn t thc y pht sinh ung th v

pht trin khi u, phn ln c tc dng liu lng thp. Cn theo cc nh khoa hc
M (Neil E. Kay v cs thuc phng th nghim Mayo Clinic) hp cht EGCG
(epigallocatechin 3- gallate) trong ch xanh c kh nng tiu dit cc t bo gy ra
bnh ung th bch cu lympho mn tnh. Kt qu th nghim cho thy, 8 trong s 10
bnh nhn ung th bch cu nguyn bo lympho mn tnh c t bo bch cu b tiu
dit di tc ng ca EGCG. Mt nghin cu khc ca cc nh khoa hc trng i
hc Rutgerbcuar (M) cho bit: khi cho polyphenol ca ch en vo mi trng
nui cy ca cc t bo rut kt bnh thng v cc t bo ung th th thy tt c cc t
bo ung th u b tiu dit trong khi cc t bo bnh thng li khng b nh hng.
Mc d mi quan h gia mc tiu dng ch v t l ngi mc bnh ung th
c nghin cu cc qun th dn c khc nhau trn th gii nhng vn cha rt
ra c kt lun r rng. Nhng hn ch c th l gii do thnh phn, hm lng
cng nh hot tnh sinh dc hc ca cc cht ha hc c sinh tng hp v tch ly
trong cy ch trng cc vng sinh thi khc nhau th khc nhau, do tc dng sinh
dc hc ca chng cng khng ng nht. Chnh v vy cc nghin cu v hm
lng, thnh phn v tc dng sinh dc hc ca cc hp cht t nhin trong cy ch
trng cc vng khc nhau vn tip tc c tin hnh.
b. Cc nghin cu trong nc.
nc ta, do v tr quan trng ca cy ch trong nn kinh t quc dn nn c
nhiu nghin cu trn i tng ny. Ch tnh t khong 10 nm tr li y c
khong vi chc cng trnh c cng b song nhng nghin cu ny tp trung ch
yu vo vic nghin cu qui trnh, iu kin trng, chm bn, chn ging, ch bin,
sinh trng pht trin v tng nng sut ca chCc nghin cu theo hng ha sinh
hc cng ch tp trung vo cc thnh phn ng vai tr chnh trong vic nng cao cht
lng ch ung. Cn cc nghin cu v ha hc v tc dng sinh dc hc ca cc
cht polyphenol khai thc t ch Vit Nam vi mc ch phc v lnh vc y dc cn
rt t; ch nhng nm gn y mi xut hin mt s cng trnh ca trng i hc Y
H Ni, i hc Khoa hc T nhin - i hc Quc gia H Ni, Vin Ha hc cc
Hp cht Thin nhin, Trung tm nghin cu v pht trin cng ngh ha sinh (thuc
Lin hip hi)... tuy nhin, cc nghin cu cn l t cha c h thng. T nm 2000
2008, nhm nghin cu ca mt s nh khoa hc (TS. o Th Kim Nhung, TS. H
Th Thanh Bnh, Ths. Th Gm) thuc Trung tm nghin cu v pht trin cng
ngh ha sinh, tin hnh kho st cc hp cht polyphenol trong l ch trng
vng Tn Cng (Thi Nguyn) trn sn phm ch Trung Du cho bit: cc hp cht
polyphenol c hot tnh sinh hc cao trong l ch ch yu l: Catechin v cc dn
xut ca catechin (catechin, epigallocatechin, gallocatechin, epigallocatechingallat,
20

epicatechin, epicatechingallat); cc cht flavonol (quercitrin, kaemferol- 3-glycozit,

kaemferol- 3-rhamnozyl, quercetagemin); theaflavin v diosmin. Hm lng
polyphenol trong l ch Tn Cng bin ng mnh theo ma v theo tui l. Bc
u nghin cu thm d v 1s hot tnh sinh hc ca ch phm Flavonoid chit xut
t l ch Tn Cng nhn thy: ch phm c kh nng chng oxy ha cao, c tc dng
bo v gan trn chut b nhim c CCl4, c tc dng khng khun tt i vi nhiu
chng vi khun gram dng v gram m, c tc dng trn vim lot. Tin hnh nghin
cu thm d trn cc t bo ung th nui cy in vitro cho thy c tc dng gy c i
vi cc t bo ung th biu m ngi Hep 2, t bo u ty chut Sp 2/0 v t bo
ung th m lin kt Sarcoma 180, nhng khng gy hy hoi cc t bo mu ngoi
vi ngi khe mnh. Mc tc dng ny ty thuc vo nng ch phm v thi
im a ch phm vo mi trng nui cy. Cc nghin cu ca Trn Th Thanh
Hng (Vin 69 B t lnh Lng) v Nguyn Th H, T Thnh Vn (B mn Ha
Ha Sinh H Y HN) [Tp ch Y Hc, 2006] cho thy: bt chit polyphenol v EGCG
ch xanh c tc dng c ch s pht trin t bo ca dng t bo ung th phi LU -1
v dng t bo ung th gan Hep G2, tc dng r nht trn dng LU 1 vi gi tr
IC50 l 3, 84 M ca EGCG ch xanh. Bc u pht hin s tng hot ca enzym
caspase 3 trn dng t bo LU -1 c b sung EGCG ch xanh vo mi trng nui
cy.

21

-Phn 3: I TNG V PHNG PHP NGHIN CU

3.1. I TNG NGHIN CU
3.1.1. i tng thc vt
a.Tin hnh iu tra v thu cc mu ch lm mu nghin cu nh sau:
+ Vng ch Thi Nguyn
(1) Ging ch Trung du l xanh (k hiu mu l TDLX)
(2) Ging ch lai LDP1 hay cn gi l ch cnh, l ging ch lai gia m l ging ch
Trung quc i Bch Tr v b l ging PH1 - ph h 1 (k hiu mu l LDP1)

nh 2: Trung Du L Xanh

nh 1: Ch Lai LDP1
+Vng ch Bo Lc Lm ng

(3) Ging ch Thanh Tm (k hiu mu l TT): dng sn xut ch Olong Thanh Tm

(4) Ging ch Kim Tuyn hay cn gi l Ngc Thu (k hiu mu l KT): dng sn
xut ch Olong Ngc Thu
(5) Ging ch Bt Tin (k hiu mu l BT): dng d sn xut ch Olong Bt Tin

nh 3: Ch Thanh Tm

nh 4: Ch Kim Tuyn
22

nh 6: Ch Thanh Tm, Bt Tin, Kim Tuyn

nh 5: Ch Bt Tin

+Vng ch Shan (Yn Bi, Mc Chu, H Giang )

(6) Ging ch Shan Cht Tin (k hiu mu l SCT)
(7) Ging ch Shan Nm Ngt (k hiu mu l SNN)
(8) Ging ch Shan Tuyt (k hiu mu l ST): l bp c lng trng nh tuyt

nh 7: Ch Shan Cht Tin

nh 8: Ch Shan Nm Ngt

Mi ging ch u c thu hi 40kg l ti

23

b.X l mu
Tt c cc mu ch thu v u c chia lm 2 phn x l theo 2 cch l: x l ti v
sy kh. Tng s mu cn x l : 8 ging ch x 2 cch x l = 16 mu
-X l mu ti: Mi mu ging ch ly 20kg l ti ra sch, loi cc l hng, l ho,
loi tp bn, sau cho ro nc. Cn xc nh trng lng, thi nh v cho vo my
xay thnh dng nhuyn, chuyn vo cc bnh ti mu, c nt nhm, th tch l 20 lt. Sau
cho cn 960 vo theo t l 1: 10. Ngm mu qua m v chit trn my siu m Power
Sonic 410 iu kin nhit 500C, thi gian 90 pht (chit mu lp li 3 ln). Gp cc
dch chit li lm dch nghin cu. o th tch tng s.
-X l mu kh: Mi mu ging ch ly 20 kg l ti ra sch, loi cc l hng, l ho,
loi tp bn, sau cho ro nc, ti ra cho kh bt, sau chuyn vo t sy. Sy
dit men trong 10 pht nhit 1100C, sau tip tc sy n kh gin 600C. Sau khi
mu kh gin th tn nh cho vo bo qun trong cc bnh ng mu c ht m. Cn
3kg mu cho vo cc bnh ng mu ti mu, c nt nhm, th tch l 20 lt. Sau cho
cn 960 vo theo t l 1: 5. Ngm mu qua m, sau chit trn my siu m Power
Sonic 410 iu kin nhit 500C, thi gian 90 pht ( chit mu lp li 3 ln)> Gp cc
dch chit li lm dch nghin cu.
-Xc nh trng lng kh tuyt i ca nguyn liu: Cn chnh xc 100g mu nguyn
liu, sy kh kit 1000C, cn xc nh n khi trng lng khng i (th nghim c
lp li 5 ln).
3.1.2 i tng ng vt
a.Mu ngi: mu ti ton phn ca ngi kho mnh (dng nhm mu O) c chng
ng bng ADC do khoa huyt hc ca bnh vin Vit - c cung cp.
b.ng vt
-Chut nht trng Swiss: do Vin V snh dch t cung cp, 6 tun tui, trng lng t 20
22 gam, trng thi sinh l bnh thng.
-Cc chng Vi khun kim nh: do ATCC cung cp
-Cc dng t bo ung th ngi: do ATCC cung cp
3.2 PHNG PHP NGHIN CU
3.2.1. Chit xut v nh lng Polyphenol tng s theo phng php ca Folin
Den`is [ 20].
-Nguyn tc: dch chit nguyn liu thu c cho phn ng vi thuc th Folin Denis
to ra sn phm c mu xanh lam. So mu trn my Corning Colorimeter 252 bc
sng = 660 nm tnh c hm lng polyphenol tng s. Cht chun l axit gallic.
-Xy dng ng chun axit Gallic: axit gallic c nng 10mg/ 50ml.
24

Bng 1: th chun axit Gallic

STT

Nng (mg)

OD

0,02

0,035

0,04

0,082

0,06

0,125

0,08

0,185

0,10

0,245

-ng chun axit Gallic:

OD
0.3
0.25
0.2
0.15
0.1
0.05
mg axit Galic

0
0

0.02

0.04

0.06

0.08

0.1

0.12

th 1 : ng chun axit Gallic

3.2.2. Chit xut v nh lng Flavonoid tng s theo phng php ca B.C. Talli [
20, 33]
a. Qui trnh chit (B.C. Talli)
Tch chit Flavonoid theo qui trnh ca B.C. Talli, cc bc trong qui trnh c
m t chi tit di y.
25

Bt nguyn liu

Chit bng clorofooc

(loi tp)

Bt nguyn liu sy kh

Dch chit clorofooc

Chit bng Ethanol

960 trn my siu m
Dch chit Ethanol
C cn p sut
Cao Ethanol
Chit bng nc ct nng
(chnh pH = 3 - 4)
Hot cht trong dch chit nc
Chit bng Ethylaxetat
Dch chit Ethylaxetat
C cn kit, p sut gim trn my ct quay

Ch phm
Flavonoid
dng bt kh

ng kh

Flavonoid
ton phn

Dng Ethanol 960

ho tan

Ch phm Flavonoid ho
tan trong Ethanol (dng
nghin cu)

Hnh 1: Qui trnh B.C. Talli

b. nh lng Flavonoid tng s
Cn chnh xc 10g bt nguyn liu, chit bng clorofooc trn my siu m; chit
trong 3 gi loi ht nha, cht bo, caroten v clorofooc. Ly phn bt nguyn liu ra,
sy cho bay ht clorofooc ri tip tc chit bng Ethanol 960 trn my siu m n khi
dch chit khng cn cho phn ng Shinoda. Dch chit Ethanol c xc nh th tch
chnh xc v bo qun trong bnh thu tinh mu nu c nt nhm kn.
Ly 50 ml dch chit Ethanol 960 em c cn iu kin p sut thp trn my ct
quay chn khng, sau chit li bng nc ct nng. Dch chit nc sau khi lc v
chnh pH = 3 4 c cho vo phu chit, dng Ethylaxetat chit nhiu ln, sau ui
26

kit dung mi trn my ct quay chn khng ti trng lng khng i. Cn xc nh

trng lng Flavonoid tng s.
Hm lng Flavonoid cha trong mu phn tch c tnh theo cng thc:

X % = [(M2 M1) x V/v x 10] / 100

Trong :
V: th tch dch chit Ethanol t 10 g bt nguyn liu
v: th tch dch chit Ethanol ly ra nh lng (50 ml)
M2: trng lng khi cn cn Flavonoid c b
M1: trng lng b
nh lng, th nghim c lp li 5 ln (n = 5), cc kt qu c x l thng k.
3.2.3. nh lng Tanin tng s theo phng php Dc in Vit Nam [ 2 ]
Tanin l hp cht kh, nn d dng b oxy ha bi KMnO4 (kalipermanganat) trong
mi trng axit vi cht ch th indigocarmin s to thnh kh cacbonic v nc, ng
thi lm mt mu xanh ca indigocarmin theo phn ng sau:
Tanin + KMnO4 CO2 + H2O
Cn c vo lng KMnO4 0,1N dng chun s xc nh c lng Tanin
tng s trong mu nghin cu.
3.2.4 Phn tch thnh phn Flavonoid bng sc k lp mng v ph t ngoi [ 10, 20]
a. Sc k lp mng
-Bn mng Silicagel F 254 ca Merk
-Chy sc k mt chiu t di ln vi h dung mi trin khai TEAF (Toluen:
Ethylaxetat: Axeton: Axit Focmic = 5: 2: 2:1).
-Quan st sc k cc iu kin khc nhau nh: nh sng t nhin; phun cc
thuc th c trng (FeCl3, NaOH, KOH, AlCl3, Willson, Diazo, Vanillin, NH4OH....);
quan st di n t ngoi bc sng 254 v 365 nm.
b. o ph t ngoi trn my Shimazu CS 9001 PC Spectrum (o ph lin tc t
bc sng 200 n 400 nm)
3.2.5 Xc nh hot peroxydaza trong mu theo E. C. Xavron [ 19]
*Nguyn tc: Mt s Flavonoid c i lc cao i vi enzym peroxydaza tham gia vo
phn ng enzym vi t cch l mt c cht v b oxy ho t dng kh (hydroquinon)
thnh dng semiquinon hoc quinon. Quinon v semiquinon sinh ra t nhng Flavonoid
tng ng l nhng gc t do bn, c kh nng trit tiu cc gc t do hot ng c hi
ca c th. Trong phn ng oxy ho Indigocarmin hot enzym cng gim th s lng
semiquinon hoc quinon c to thnh cng nhiu v iu th hin kh nng chng
oxy ho ca Flavonoid. Trong phn ng ny, Flavonoid ng vai tr l c cht cnh tranh
vi Indigocarmin. Da vo vic xc nh cng mu ca phn ng oxy ho c cht
27

indigocarmin bi H2O2 trong mi trng axit yu khi c s tham gia ca enzym

peroxydaza bc sng 610 nm c th tnh c hot tnh enzym ( v t s tnh c
% c ch hot ng ca enzym). Hot tnh enzym peroxydaza mu i chng (khng c
mu th) l 100%, khi c cht th hot tnh enzym s thay i.
*Phng php th hot tnh chng oxy ha (xc nh hot peroxydaza mu ngi)
+Nguyn liu th nghim:
- Mu ti c chng ng bng ADC (Citrate dextrose adenine), pha long 500 ln
bng dung dch NaCl 0,9%. Th nghim c tin hnh trn nhm mu O ca ngi khe
mnh.
- Cht th l cc ch phm Flavonoid chit xut t cc mu l ch khc nhau. Cht th
c pha trong DMSO 100% sao cho t c cc nng l: 128 g/ml; 32g/ml;
8g/ml; 2g/ml; 0,5 g/ml v nng DMSO trong phn ng khng ln hn 1%.
+Phn ng: 60 l m axetat natri pH 4,7 0,1N
60 l pha long 500 ln
60 l H2O2 0,2N
20 l Indigocarmin 0,001N
+ thi gian phn ng 20 pht, sau c kt qu bc sng 610 nm, trn my
quang ph TECAN v x l s liu bng phn mm raw data.
3.2.6. Nghin cu tc dng khng khun ca cc ch phm Flavonoid chit xut t
cc mu l ch khc nhau [ 6, 25, 31 ].
Cc th nghim c tin hnh theo phng php pha long a nng .
* Mi trng nui cy:
MBH (Mueller Hinton Broth), MHA (Mueller Hinton Agar), TSB (Tryptic Soy
Broth), TSA (Tryptic Soy Agar) cho vi khun; SAB, SA cho nm.
*Vi sinh vt: Th nghim s dng 7 chng vi sinh vt kim nh v nm i din gy bnh
ngi l:
+ Vi khun Gram (-): Pseudomonas aeruginosa (Pa) ATCC 15442; E. Coli (Ec) ATCC
25922; Salmonella enterica.
+ Vi khun Gram (+): Staphylococcus aureus (Sa) ATCC 13709; Bacillus subtilis (Bs)
ATCC 6633; Lactobasillus fermentum N4.
+ Nm Candida albicans (Ca) ATCC 10231.
*Nng cc ch phm:
Cc ch phm Flavonoid chit xut t cc mu l ch khc nhau c pha long nh sau:
ch phm ban u c pha trong DMSO c nng l 40 mg/ml, sau s pha long
28

thnh cc nng khc nhau nh: 256 g/ ml;156 g/ ml; 64 g/ml; 16 g/ ml; 4g/ml;
1g/ml th hot tnh vi cc chng vi sinh vt kim nh trn.
- Tin hnh: chun b dung dch vi sinh vt hoc nm vi nng 5. 105, ly 10 l dung
dch mu th theo cc nng c pha long thm 200 l dung dch vi sinh vt hoc
nm. 370 C sau 24 gi c gi tr MIC bng mt thng sau khi b sung 50 l MTT
(1mg/ml) vo cc ging th, gi tr MIC c xc nh ti ging c nng cht th tho
nht khng chuyn ho MTT thnh MTT formazan cho mu tm m. IC 50 c tnh
ton da trn s liu c c t bo bng my Tecan (Genios) v phn mm tnh gi tr
IC 50 Raw data program.
3.2.7. Nghin cu tc dng c t bo invitro ca cc ch phm Flavonoid chit xut
t cc mu l ch khc nhau [14, 27 ].

*Nguyn vt liu th nghim:

-Mu th: cc ch phm Flavonoid chit xut t cc mu l ch khc nhau
-Cc dng t bo ung th: KB (Human epidermic carcinoma), HepG2 (Hepatocellular
carcinoma); Lu (Human lung carcinoma), MCF -7 (Human breast carcinoma), S -180
(Sarcoma 180).
-Ha cht: MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) ca
hng in vitrogen, DMSO ca hng sigma v cc ha cht khc
-My mc thit b: my c ELISA ca hng Thermo Labsystems
* Phng php:
+Nguyn tc:
p dng phng php MTT ca Mosmann (1983) nh gi c tnh trn t bo.
Nguyn tc ca phng php nh sau: MTT l th nghim in vitro o s tng sinh v
sng st ca t bo. T bo c nui cy trong mt a 96 ging, y bng. Hp cht
MTT 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide, c mu vng c
thm vo mi ging v t bo c 37oC, 5% CO2. Mu vng ny b bin i thnh
formazan tm trong ty th ca nhng t bo sng. Kh nng hp th ca dung dich c mu
ny c th c nh lng bng my quang ph k bc song 540 600 nm. S bin
i mu ch xy ra khi enzyme reductase trong ty th l hot ng, v do s chuyn
i c th lin quan trc tip n s lng t bo sng st.
+ Cch tin hnh:
-Nui cy t bo: Cc dng t bo c lu gi trong nit lng, nh thc v duy tr trong
mi trng DMEM (Dulbecco's Modified Eagle Medium) c b sung huyt thanh b ti
10%, dung dch khng sinh v khng nm 1% (penicillin 50,000 units/L v streptomycin

29

50 mg/L). T bo c nui cy cho pht trin ti mc khong 70%, thay mi trng

sch, t bo ny c dng lm th nghim.
- Mu th nghim: Ha mu th nghim vo dung dch DMSO 100% sao cho nng gc
ca cc mu l 2 mg/ml. Pha cc ch phm th thnh thang nng gm 5 nng : 0,5
g/ml; 2 g/ml; 8 g/ml; 32 g/ml; 128 g/ml. Ellipticin c s dng lm chng dng.
Dung dch DMSO 0,1% c s dng lm chng m.
- Quy trnh nui cy:
.Cho khong 5000 t bo vo 200 l mi trng nui cy/ 1 ging ca a nui cy
96 ging. 2 ging trng lm chng trng (blank control).
.Cc t bo c nui trong mi trng 37oC v 5% CO2 cho php t bo gn vo
y mi ging ca a nui cy. Gi t bo trong 24 gi t bo n nh.
.Sau 24 gi nui cy, thm 10 l thuc th cc nng khc nhau vo mi ging.
Mi nng c lp li 2 ging. Lc nh a nui cy thuc c tan hon ton
trong mi trng.
. a nui cy t 48 gi (37oC, 5% CO2) cho php thuc pht huy tc dng. Quan
st t bo hng ngy bng knh hin vi.
.Chun b dung dch MTT nng 5mg/ml.
.Thm 20 l dung dch MTT vo mi ging ca a nui cy. Lc nh cho MTT
khuch tn u trong mi trng nui cy.
. 37oC trong 4 gi MTT c chuyn ha.
.Loi b mi trng trong cc ging ca a nui cy.
.Hon tr formazan (sn phm chuyn ha MTT) bng 100 l DMSO. Lc k
formazan c th tan hon ton.
.c mt quang bc sng 540 nm trn my Spectrophotometter Genios
TECAN. Mt quang s phn nh s lng t bo sng st.
- % t bo sng sau khi x l thuc so vi chng trng (t bo khng c x l bng
thuc c gi l % t bo sng, c tnh theo cng thc nh sau:
% t bo sng = [OD540 ca t bo c x l] x 100%/ [OD540 ca t bo khng c x l
thuc]

-Cch tnh gi tr IC50: Gi tr IC50 c tnh bng phn tch hi quy khng tuyn tnh
ca ng cong p ng liu tng ng, s dng phn mm Graphpad Prism 5.0
3.2.8 Xc nh c tnh cp theo phng php ca A. Wallace Hayes [34 ]
-Da theo nguyn tc v phng php th c tnh cp ca Wallace Hayes.
-i tng ng vt l chut nht trng chng Swiss, trng lng 20 2 g. Chut c
nhn n 15 gi trc khi th nghim, ung nc theo nhu cu. Kim tra cn nng trc khi
th nghim. Chut t cc yu cu v cn nng c a vo th nghim.
30

-Mu th l cc ch phm Flavonoid chit xut t cc mu l ch khc nhau. Mu th

c pha thnh cc dung dch c nng tng dn ri cho chut ung.
-a mu th di dng hn dch theo ng ung. Ly th tch mu th theo qui nh
a thng vo d dy chut bng kim cong u t. Da trn kt qu thm d trong th
nghim s b, tin hnh th nghim chnh thc trn 36 chut, chia thnh 6 nhm gm 1
nhm chng dng nc ct v 5 nhm th theo mc liu d tnh.
Nhm 1 (nhm i chng): cho ung nc ct
Nhm 2: ung 0,15ml mu th /20g trng lng/ngy (tng ng 187,5 mg/kg)
Nhm 3: ung 0,35ml mu th /20g trng lng/ngy (tng ng 437,5 mg/kg)
Nhm 4: ung 0,40ml mu th /20g trng lng/ngy (tng ng 500 mg/kg)
Nhm 5: ung 0,5ml mu th /20g trng lng/ngy (tng ng 625 mg/kg)
Nhm 6: ung 0,80ml mu th /20g trng lng/ngy (tng ng 1000 mg/kg)

-Lch theo di: theo di biu hin ca chut sau khi ung trong 24 gi u v theo di
hot ng ca ng vt th nghim trong thi gian 7 ngy sau khi ung.
3.2.9 Nghin cu tc dng bo v gan ca ch phm Flavonoid chit xut t ch
Trung Du l xanh F15 (in vivo) [ 6, 11,16 ]
+Nguyn tc:
Gy tn thng gan chut thc nghim bng cch tim i.p CCl4 vi liu 4ml/kg. Sau
cho chut nht trng ung mu th theo cc liu d nh. nh gi tc dng bo v gan
ca cc ch phm cn th qua vic kho st kh nng c ch qu trnh peroxy ho lipid
(POL). Cc gc t do ca oxy sinh ra trong gan chut l nhng tc nhn chnh gy ra
phn ng peroxy ha cc cht hu c (ch yu l cc axit bo cha no mng t bo);
mt trong nhng sn phm ca qu trnh ny l dialdehyt malonyl (DAM). Cht ny
(DAM) phn ng vi axit thiobarbituric to ra phc c mu hng bn =532 nm. o
cng mu ca phc (OD) c th bit lng DAM sinh ra nhiu hay t. Hot tnh
chng oxy ha (HTCO) l t l % lng DAM gim i mu th so vi mu i chng.
Th nghim c tin hnh vi cc nng Flavonoid khc nhau.
+M hnh th nghim: th nghim c tin hnh trn chut v c chia thnh 3 l
- L 1: l i chng sinh hc (CSH): chut c nui v chm sc bnh thng
khng dng ch phm.
- L 2: chut c tim 0,2 ml dung dch CCl4 10% trong du vng, chm sc
bnh thng.
- L 3: chut va c tim 0,2 ml dung dch CCl4 10% trong du vng va c
cho ung mu th F15.
31

Sau 72 gi tt c cc chut th nghim u b git ly gan quan st tn thng

thc th ca gan v xc nh DAM theo phng php C.G.Blagodorov:
+Ho cht: m Tris, FeSO4, axit ascorbic, TCA, axit thiobacbituric...
+Tin hnh: M ly gan chut nht. Cn chnh xc 100mg gan chut lm homogennat
trong ci s iu kin 40 C vi 5 ml m Tris 0,04 M, pH = 7,4 v 10 ml H2O. Ly tm
600 vng / pht trong 10 pht. Sau ly 3 ml dch ly tm cho vo hn hp gm: 0,4 ml
FeSO4 10-3 M; 0,4 ml dung dch axit ascorbic 10-2 M. hn hp 370 C trong 1 gi, ly
hn hp ra. Thm 2 ml dung dch axit thiobacbituric 0,25%. un si cch thu khong
15 pht, ngui. o mu bc sng 532 nm.
+Lng DAM c tnh theo cng thc:
C = D/ . l
Trong :
C: s mol DAM

D: mt quang hc

l: chiu dy cuvet

: h s tt mol = 1,56.10-5 M cm-1

3.2.10. Nghin cu tc dng h lipid huyt ca ch phm Flavonoid F15 (chit xut t
ch Trung Du l xanh) trn m hnh tng lipid huyt ni sinh gy bi Triton WR 1339 trn chut nht trng [ 6 ].
*Nguyn vt liu
-ng vt th nghim: chut nht trng trng lng t 23-27 g, c c v ci t tiu
chun th nghim.
-Ha cht
.Cht gy tng lipid: Triton WR 1339 ca Sigma,
.Kt nh lng Cholesterol, triglycerid ca Human
-Mu th:
.Ch phm Flavonoid chit xut t ch Trung Du l xanh, dng vi liu tng ng
250 mg/kg th trng chut
Thuc i chiu Fenofibrate (bit dc Lipanthyl ca Php hm lng 100 mg): liu
100 mg/kg th trng chut
-My mc thit b: my nh lng sinh ha bn t ng Scout ca
*Phng php nghin cu
-S dng m hnh gy tng lipid huyt ni sinh bng triton WR 1339 ca Frantz ID v cs,
c ci tin, Triton WR 1339 c ch s thanh thi cc lipoprotein giu triglycerid gy ra s
tng lipid huyt cp tnh, thi gian gy tng lipid huyt ti a l khong 18-20 gi sau khi
tim, sau n dn tr v mc ban u,
32

-M hnh c tm tt nh sau: Chia chut lm 3 l

+ L C: chng bnh l
+ L F15: ung F15 liu tng ng 250 mg/kg th trng chut
+ L Feno: ung fenofibrat liu tng ng 100 mg/kg
-Ly mu ui chut nh lng cholesterol v triglycerid huyt thanh (thi im ban u
- to). Cho chut ung thuc (cc l th thuc) hoc nc (l chng bnh l) ngy th nht
v ngy th 2, ngy th 3, sau khi cho chut ung 30 pht, tim tnh mch ui chut
dung dch Triton vi liu tng ng 250 mg/kg th trng chut. Sau 17 gi tim Triton,
cho chut ung thuc hoc nc, 1 gi sau ung, ly mu nh lng cholesterol v
triglycerid huyt thanh - thi im ts)
-Phng php nh gi kt qu: Tnh % tng hm lng cholesterol ton phn v
triglycerid huyt thanh ca cng l th nghim trc v sau khi tim Triton, so snh mc
tng ca cc l th thuc so vi l chng bnh l. Cc s liu c biu th bng s
trung bnh MSE v x l thng k theo Student t-test vi P < 0,05.
3.2.11. Nghin cu hot tnh chng ung th trn ng vt thc nghim [ 28]
-Nguyn tc: dng dng t bo S -180 vi s lng 5 x 106 t bo tim vo i chut.
Cc t bo ung th S 180 s pht trin m lin kt xen gia m c. iu tr chut
mang ung th bng ch phm F14 v nh gi kh nng hn ch s pht trin ca t bo
ung th ca ch phm F14 bng ch s Growth Delay (GD)
-i tng ng vt: chut nht khe mnh BAL b/c trng lng t 20 -25g, s lng 20
con chia 2 l: 1 l i chng v 1 l th nghim. l th nghim, cho chut ung ch
phm F14 hng ngy vi liu 5 mg/con (tng ng vi 2 ml/con) k t khi tim t bo ung
th n ngy th 15 m chut. L i chng cho chut ung nc ct thay cho ch phm
F14 vi th tch tng ng.
-nh gi tc dng ca thuc qua cc ch tiu:
.Th tch khi u: dng thc kp o kch thc di, rng ca i chut c tim t
bo ung th cc thi im 4, 6, 10, 13, 15 ngy sau khi tim. T s trn tnh th tch
khi u theo cng thc:
V = x D3/6 (theo Nowark, 1978)
.Tnh t l phn trm hn ch pht trin khi u:
Ch s GD: (Growth Delay) = (Vc Vt) x 100/Vc
Trong :
Vc: Th tch khi u l chng
Vt: Th tch khi u l th nghim
.Tnh trng lng tng i ca u i: git chut ngy th 15, ly trng lng
i mang u tr i trng lng i bn i din.
33

.Kh nng hn ch di cn t bo ung th t u i ln gan v phi: tc dng hn ch

di cn c nh gi thng qua vic m nt di cn bng knh lp.
3.2.12 X l s liu theo phng php thng k sinh hc [ 9]
Tr s trung bnh: X =

Xi
n

( X i X )2
lch chun: S =
(n 1)
S
Sai s chun: S x =
n

Khong trung bnh: ( X t ( ,n 1)

X
(n 1)

Trong :
X : S trung bnh cng
X i : Gi tr tng s liu

n: S ln th nghim
Ngoi ra c s dng cc phn mm x l s liu nh: Graphpad Prism 5.0; phn mm
tnh gi tr IC50 Raw data program....

34

Phn 4: KT QU NGHIN CU
-i tng nghin cu l 8 ging ch thuc 3 vng sn xut ch:
* Vng Tn Cng Thi Nguyn:
-Ging ch Trung Du l xanh (k hiu TDLX)
-Ging ch lai LDP1 (k hiu l LDP1)
y l nhng ging ch ung truyn thng ca ng bo min Bc (vng ng
bng) c ch bin thnh ch xanh hoc ch en.
*Vng ch Bo Lc Lm ng
-Ging ch Thanh Tm (k hiu l TT)
-Ging ch Kim Tuyn (k hiu KT)
-Ging ch Bt Tin (k hiu BT)
y l nhng ging ch c di thc t i Loan sang Vit Nam, c trng
ch yu Lm ng v dng ch bin thnh ch Olong.
*Vng ch Shan - Mc Chu
-Ging ch Shan Cht Tin
-Ging ch Shan Nm Ngt
-Ging ch Shan Tuyt
y l cc ging ch trng trn i ni cao, ni c kh hu mt. Cy ch c tui
th 25 nm, ring ch Shan Tuyt c bo tn nh nhng cy c th, c chiu cao
v tn l pht trin t nhin, t c chm bn v khng phun cc loi thuc bo v
thc vt. Cc loi ch Shan c th c ch bin thnh ch xanh kh theo cng ngh
truyn thng hoc thnh ch en.
-B phn s dng: l bnh t
-Thi gian thu mu: thng 3 - 6
-Kt qu nghin cu gm 2 phn chnh: nghin cu v ha hc v nghin cu tc dng
sinh hc.

4.1. CC NGHIN CU V HO HC
Hng trm cng trnh nghin cu v cy ch (Camellia sinensis) ca nhiu tc
gi trn th gii khng nh cc hp cht Polyphenol ch mang li nhiu li ch cho
sc khe ca ngi s dng , trong nhm cht Flavonoid (bao gm ch yu cc dn
xut Catechin v mt phn nh Flavonoid khc) c coi nh nhng hot cht ch yu
trong ch.
Vit Nam, trong nhng nm gn y ngoi ging ch truyn thng c
trng v s dng lu i l ch TDLX cn c nhiu ging ch khc nh: ch lai
35

LDP1; cc ging ch di thc t i Loan ti (Thanh Tm, Kim Tuyn, Bt Tin);

cc ging ch Shan mc trn vng cao nh Sui Ging, Mc Chu, H Giang
V vy, c th khai thc gi tr cy ch Vit Nam nh mt cy dc liu
cung cp ngun hot cht Flavonoid dng cho mc ch Bo v sc khe, cn c
nhng nghin cu thm d. Di y l mt s kt qu v ha hc lin quan n nh
lng, nh tnh cc cht Polyphenol v phng thc x l mu nguyn liu i vi 8
ging ch k trn.
4.1.1. Kho st s c mt ca nhm cht Polyphenol bng cc phn ng nh tnh
c trng
T cc dch chit cn chng ti tin hnh kim tra cc phn ng nh tnh c
trng ca nhm cht Polyphenol kt qu c trnh by bng 2
Bng 2: Cc phn ng nh tnh ca cc dch chit thu c
Tn mu

K hiu
mu

P/u
NaOH 10%

P/u
FeCl3 5%

P/u
Shinoda

P/u
Diazo

P/u
Vanillin

Trung du l xanh

TDLX

Vng nu 4+

Xanh en 4+

4+

Vng cam 4+

son 4+

Ch lai LDP1

LDP1

Vng nu 4+

Xanh en 4+

4+

Vng cam 4+

son 4+

Thanh Tm

TT

Vng nu 4+

Xanh en 4+

4+

Vng cam 4+

son 4+

Kim Tuyn

KT

Vng nu 4+

Xanh en 4+

3+

Vng cam 3+

son 4+

Bt Tin

BT

Vng nu 4+

Xanh en 4+

4+

Vng cam 4+

son 4+

Shan Cht Tin

SCT

Vng nu 4+

Xanh en 4+

nu 3+

Vng cam 3+

son 2+

Shan Nm Ngt

SNN

Vng nu 4+

Xanh en 4+

4+

Vng cam 4+

son 4+

TS

Vng nu 4+

Xanh en 4+

nu 2+

Vng cam 3+

son 2+

Shan Tuyt

Ghi ch:
Du +: ch phn ng dng tnh
Cc con s: ch mc m nht ca mu phn ng
Cc kt qu thu c bng 2 cho thy cc phn ng nh tnh c trng ca nhm
cht Polyphenol rt in hnh:
-Phn ng FeCl3 cho mu xanh en c trng cho thy s c mt ca nhng
Polyphenol c cu to orthodiphenol
-Phn ng Shinoda c mu m c v phn ng Diazo c mu vng cam in hnh
ch ra s c mt cc cht Flavonoid mang nhm cacbonyl (>C = 0) v tr C4; c
im cu to ho hc ny l mt trong nhng yu t c bn cn c i vi mt Bio
Flavonoid (cc cht Flavonoid c hot tnh sinh hc).
-Phn ng Vanillin 1% c mu son c trng cho thy trong cc mu l ch ny c
cha catechin v cc dn xut ca catechin.

Qua cc kt qu thu c cho thy tt c 8 ging ch nghin cu u giu

Polyphenol, c bit l nhm hp cht Flavonoid.
4.1.2. Xc nh trng lng kh tuyt i ca l ch
36

T cc mu l ch mi thu hi, chng ti tin hnh x l mu nguyn liu theo

cc qui nh chung v xc nh trng lng kh tuyt i (TLKT) ca nguyn liu
nh sau: cn chnh xc 100g mu nguyn liu, sy kh kit 1000C, sau cn v ghi
li trng lng (cn n khi trng lng khng i). Cc kt qu thu c trnh by
bng 3 (th nghim c lp li 5 ln).
Bng 3: Trng lng kh tuyt i ca l ch
TT

Tn mu

K hiu
mu

% Trng lng
kh tuyt i

Trung du l xanh

TDLX

34,0

Ch lai LDP1

LDP1

35,0

Thanh Tm

TT

32,0

Kim Tuyn

KT

45,0

Bt Tin

BT

45,0

Shan Cht Tin

SCT

42,0

Shan Nm Ngt

SNN

44,0

Shan Tuyt

ST

39,0

Nh vy, hm lng cht kh cao nht l trong l ch Kim Tuyn = Bt Tin

v thp nht l l ch Thanh Tm; hay ni theo cch khc th hm lng nc trong
cc ging ch gim dn theo th t sau:
Kim Tuyn = Bt Tin < Shan Nm Ngt < Shan Cht Tin < Shan Tuyt <
Ch lai LDP1 < Trung Du l xanh < Thanh Tm.
4.1.3. nh lng Polyphenol tng s trong l ch
Tin hnh nh lng Polyphenol tng s ca 16 mu ch (8 mu ti v 8 mu
kh) theo phng php Folin Denis. Kt qu c tnh theo % trng lng kh
(TLK) i vi mu kh; tnh theo % trng lng ti (TLT) i vi mu ti v %
quy i t mu ti thnh kh theo trng lng kh tuyt i (TLKT).
Bng 4: Hm lng Polyphenol tng s trong l ch
TT

Tn mu

K hiu
mu

% Hm lng
Polyphenol tng
s theo TLT

% Hm lng
Polyphenol tng s theo
TLK (thc t thu c)

% Hm lng
Polyphenol tng s
qui theo TLKT

Trung du l xanh

TDLX

5,76 0,12

16,58 0,69

16,94

Ch lai LDP1

LDP1

5,41 0,31

15,60 0,17

15,45

Thanh Tm

TT

6,15 0,24

17,01 0,29

19,21

Kim Tuyn

KT

6,52 0,17

13,98 0,12

14,48

Bt Tin

BT

10,51 0,62

18,02 0,15

23,35

Shan Cht Tin

SCT

11,15 0,13

24,40 0,16

26,54

Shan Nm Ngt

SNN

9,19 0,23

19,32 0,10

21,88

Shan Tuyt

ST

6,89 0,11

15,21 0,25

17,66

37

Kt qu bng 4 v biu 2a, 2b, 2c cho thy hm lng Polyphenol tng s ca

cc mu ch u chim mt t l tng i cao:
- cc mu ti: hm lng Polyphenol chim t 5,41% 11,15 %; hm lng cao
nht l mu Shan Cht Tin (11,15%) v thp nht l mu ch lai LDP1 (5,41%).
- cc mu kh:
.Hm lng Polyphenol chim t 13,98% - 24,40%; hm lng Polyphenol cao
nht l mu Shan Cht Tin (= 24,40%) v thp nht l mu ch Shan Tuyt
(=15,21%) v ch lai LDP1 (= 15,60%).
.Trong nhm ch vng Tn Cng Thi Nguyn hm lng Polyphenol tng
theo th t sau: ch Trung Du l xanh > ch Lai LDP1.
.Trong nhm ch Shan th hm lng Polyphenol tng theo th t sau: ch Shan
Cht Tin > ch Shan Nm Ngt > ch Shan Tuyt.
.Trong nhm ch vng Bo Lc - Lm ng hm lng Polyphenol tng theo
th t sau: ch Bt Tin > ch Thanh Tm > ch Kim Tuyn.
Cc kt qu thu c cho thy y l nhng nguyn liu giu Polyphenol c th
hng ti khai thc ng dng vo thc tin.
% Hm lng
Polyphenol

TLT
% Hm lng
Polyphenol

20

15.45

16.58

23.35

25

TLK

16.94

20

15.6

17.01

15

15

TLK
14.48

5.41

TLK

21.88

TLK

24.4

TLQ

19.32

20

17.66

Ghi ch:

15.21

15
11.15

9.19

10

6.89

TLQ
TLK

0
1

TLT

TLT

26.54

30

Biu 2b: Hm lng Polyphenol tng s ca ch Bo Lc Lm ng

1: ch Thanh Tm
2: ch Kim Tuyn
3: Bt Tin

Biu 2a: Hm lng Polyphenol tng s ca ch Tn Cng Thi Nguyn

1: ch TDLX
2: ch lai LDP1

25

TLQ
TLK

% Hm lng
Polyphenol

6.52

TLT

10.51

6.15

TLQ

TLQ

18.02

13.98

10

10
5.76

TLT

19.21

TLQ

TLT

Biu 2c: Hm lng Polyphenol tng s ca ch Shan

1: ch Shan Nm Ngt
2: ch Shan Cht Tin 3: ch Shan Tuyt

38

TLT: % Polyphenol tnh theo

trng lng ti
TLK: % Polyphenol tnh theo
trng lng kh
TLQ: % Polyphenol tnh theo
qui i theo TLKT

4.1.4. nh lng Tanin tng s trong l ch

T tt c cc mu nguyn liu thu c chng ti tin hnh nh lng Tanin
tng s (% theo TLK) theo phng php trong Dc in Vit Nam, cc kt thu c
trnh by biu 3:
Theo TLT
% Hm lng
Tanin
20

Theo TLK
18.1

17.35

17.85
14.68

15

10

8.1

7.7

6.81

6.75

15.4

6.74

14.86
13.85

13.54

7.53

6.81

6.92

Ghi ch:
1.Trung du l xanh
2.Ch lai LDP1
3.Thanh Tm
4.Kim Tuyn
5.Bt Tin
6.Shan Cht Tin
7.Shan Nm Ngt
8.Shan Tuyt

Tn mu

0
1

Biu 3: Hm lng Tanin tng s trong l ch

Kt qu bng 1 (phn ph lc) v biu 3 cho thy:

+ Hm lng Tanin tng s ca cc mu ch u chim mt t l tng i cao:
- cc mu ti hm lng Tanin chim t 6,74 8,10 %; cc mu kh hm lng
Tanin chim t 14,86% - 18,52%.
- Hm lng Tanin cao nht mu ch Trung Du l xanh v thp nht mu ch Bt
Tin
+ Theo kt qa nghin cu ca Vin nghin cu ch cho bit : ch Ph Th 1 tm 3
l c hm lng Tanin l 30,3% (TLK) [12,13]; cn theo tc gi Th Gm (
Nghin cu mt s c im ha hc v tc dng sinh hc ca cc cht thc vt th
sinh trong cy ch- lun n tt nghip Thc s Sinh Hc)[7]; Ch Trung du vng
Tn Cng hm lng Tanin nh sau: trong l bp (1 tm 2 l) l 30,107 0,53 (%
theo TLK); ch bp kh thnh phm l: 20,73 0,41 (% theo TLK); l bnh t l 15
0,11 (% TLK). Cn theo mt s nghin cu khc cho bit: Tanin trong ch en (ch
b ln men) cc nc khc c hm lng trung bnh l 15% 18% (% theo TLK),
cn trong ch xanh thnh phm (ch c ch bin t l bp non v cha b ln men)
cng ch c t 20 30% (% theo TLK)
Nh vy, khi so snh gia kt qu thu c ca ti vi cc cng trnh cng
b khc cho thy hm lng Tanin ca 8 ging ch nghin cu u chim mt t l
tng i cao.

4.1.5. Chit xut v nh lng Flavonoid tng s trong l ch

Tin hnh chit xut v nh lng Flavonoid tng s theo qui trnh B.C.Talli,
cc kt thu c trnh by bng 5 v biu 4a, 4b, 4c
39

Bng 5: Hm lng Flavonoid tng s trong l ch

TT

Tn mu

K hiu
mu

% Hm lng
Flavonoid tng
s theo TLT

% Hm lng
Flavonoid tng s theo
TLK (thc t thu c)

% Hm lng
Flavonoid tng s
qui i theo TLKT

1
2
3
4
5
6
7
8

Trung du l xanh
Ch lai LDP1
Thanh Tm
Kim Tuyn
Bt Tin
Shan Cht Tin
Shan Nm Ngt
Shan Tuyt

TDLX
LDP1
TT
KT
BT
SCT
SNN
TS

2,23 0,21
3,01 0,32
2,34 0,11
3,10 0,24
3,04 0,15
1,67 0,10
1,95 0,12
1,82 0,14

5,77 0,36
5,04 0,10

6,55
8,60
7,31
6,88

% Hm lng
Flavonoid

TLT

TLQ

7
5

3.01

2.23

3.1

3.04

2.34

4.54

4.48

TLQ

5.47

5.04

6.75

6.88

5.77

TLK
7.31

6.55

6,75
3,97
4,43
4.55
TLT

% Hm lng
Flavonoid
8

TLK

8.6

4,48 0,21
4,54 0,25
5,47 0,12
2,93 0,10
3,29 0,23
2,51 0,11

1
2

Biu 4a: Hm lng Flavonoid trong l ch Tn Cng Thi Nguyn

1: Ch Trung Du l xanh
2: Ch lai LDP1
% Hm lng
Flavonoid

TLQ

3.29

3.5

TLT
TLK

3.97

4
2.93

2.51

2.5
2

4.55

4.43

4.5

Biu 4b: Hm lng Flavonoid trong l ch Bo Lc Lm ng

1: Ch Thanh Tm
2: Kim Tuyn
3: Bt Tin

1.95

1.67

1.82

1.5
1
0.5
0
1

Biu 4c: Hm lng Flavonoid trong l ch Shan

1: Ch Shan Cht Tin 2: Ch Shan Nm Ngt 3: Ch Shan Tuyt

Kt qu bng 5 v biu 4a, 4b, 4c cho thy hm lng Flavonoid tng s ca cc

mu ch u chim mt t l tng i cao:

40

- cc mu ti: Hm lng Flavonoid chim t 1,82% 3,04 %; hm lng cao

nht l mu Thanh Tm (3,34 %) v thp nht l mu ch Shan Tuyt (1,82 %).
- cc mu kh:
.Hm lng Flavonoid chim t 2,51% - 5,77%; hm lng Flavonoid cao nht
l mu Trung du l xanh (= 5,77 %) v thp nht l mu ch Shan Tuyt
(=2,51%):
.Trong nhm ch vng Tn Cng Thi Nguyn hm lng Flavonoid tng
theo th t sau: ch Trung Du l xanh > ch Lai LDP1.
.Trong nhm ch vng ch Shan th hm lng Flavonoid tng theo th t sau:
ch Shan Nm Ngt > Shan Cht Tin > Shan Tuyt.
.Trong nhm ch vng Lm ng hm lng Polyphenol tng theo th t sau:
ch Bt Tin> Kim Tuyn> Thanh Tm.
Khi so snh gia hm lng Polyphenol, Tanin v Flavonoid cc mu ti c

qui i theo trng lng kh vi hm lng Polyphenol, Tanin, Flavonoid thc t thu
c ca mu kh cho thy: trong qu trnh lm kh mu (l), hm lng cc cht
mu nguyn liu kh c b mt i so vi nguyn liu ti; song vic thu gom bo qun
mt khi lng nguyn liu ti l rt kh thc thi nn ta buc phi s dng nguyn
liu kh nu thnh phn v hot tnh sinh hc ca cc hot cht khng b thay i.

4.1.6. Phn tch thnh phn Flavonoid tng s thu c bng sc k lp

mng v ph t ngoi.
* Vic phn tch thnh phn Flavonoid tng s ca cc mu bng sc k lp mng v
ph t ngoi c tin hnh ng thi trn 20 ch phm Flavonoid thu c:
-Gm 8 ch phm Flavonoid chit xut t cc mu ti, c k hiu mu nh sau:
F1: Ch phm Flavonoid ca ging ch Thanh Tm, l ti
F2: Ch phm Flavonoid ca ging ch Bt Tin, l ti
F3: Ch phm Flavonoid ca ging ch Kim Tuyn, l ti
F4: Ch phm Flavonoid ca ging ch Shan Nm Ngt, l ti
F5: Ch phm Flavonoid ca ging ch Shan Cht Tin, l ti
F6: Ch phm Flavonoid ca ging ch Shan Tuyt, l ti
F7: Ch phm Flavonoid ca ging ch Trung Du l xanh, l ti
F8: Ch phm Flavonoid ca ging ch lai LDP1, l ti
-V 8 ch phm Flavonoid chit xut t cc mu kh, c k hiu mu nh sau:
F9: Ch phm Flavonoid ca ging ch Thanh Tm, l kh
F10: Ch phm Flavonoid ca ging ch Bt Tin, l kh
F11: Ch phm Flavonoid ca ging ch Kim Tuyn, l kh
F12: Ch phm Flavonoid ca ging ch Shan Nm Ngt, l kh
F13: Ch phm Flavonoid ca ging ch Shan Cht Tin, l kh
F14: Ch phm Flavonoid ca ging ch Shan Tuyt, l kh
F15: Ch phm Flavonoid ca ging ch Trung Du l xanh, l kh
F16: Ch phm Flavonoid ca ging ch lai LDP1, l kh
* phn tch thnh phn Flavonoid trn bn mng chng ti s dng nhiu phng
php phi hp:
41

- Sc k lp mng mt chiu: c tin hnh trn bn mng Silicagel mi mu c

chm ln 5 bn (lp li 5 ln ); chy sc k mt chiu di ln vi h dung mi TEAF.
Sau khi chy sc k xong, bn mng c lm kh trong t m nhit 32o 37o ;
sau ly ra v x l vi cc thuc th c trng quan st sc k trong nhng
iu kin: nh sng t nhin; di n t ngoi bc sng 254 v 365 nm; phun
cc thuc th c hiu ca nhm cht Flavonoid: FeCl3, NaOH, KOH, AlCl3, Willson,
Diazo, Vanillin, NH4OH....
- o ph t ngoi trn my Shimazu CS 9001 PC Spectrum (o ph lin tc t
bc sng 200 n 400 nm)
* Cc kt qu thu c trnh by bng 6, nh 9, 10, 11 v hnh 2

Ghi ch:
Theo th t t tri sang phi

TT: Thanh Tm
BT: Bt Tin
KT: Kim Tuyn
SNN: Shan Nm Ngt
SCT: Shan Cht Tin
TSMC: Shan Tuyt
TDLX: Trung Du l Xanh
LDP1: ch lai LDP1
TT

BT

KT

SNN

SCT

TSMC

TDLX

TDLX

nh 9: Sc k ca cc ch phm Flavonoid ch
(quan st iu kin nh sng thng)

LDP1

Ghi ch:
TT: Thanh Tm
BT: Bt Tin
KT: Kim Tuyn
SNN: Shan Nm Ngt
SCT: Shan Cht Tin
TSMC: Shan Tuyt
TDLX: Trung Du l Xanh
LDP1: ch lai LDP1
TT BT

KT

SNN SCT TSMC TDLX TDLX LDP1 LDP1

nh 10: Sc k ca cc ch phm Flavonoid ch khi phun thuc th Diazo

42

Ghi ch:
1: Trung Du l Xanh
2: Shan Cht Tin
3: Bt Tin

nh 11: Sc k ca cc ch phm Flavonoid ch khi

phun thuc th Vanillin
Rf

0,74
0,70
0,64
0,56
0,47
0,43
0,39
0,36
0,27
0,23
0,18

TT

BT

KT

SCT

SNN

TS

TDLX

LDP1

Hnh 2 : Sc k ca cc ch phm Flavonoid thu c t l ch

43

Bng 6: c im cc vt tch ra trn sc k ca cc ch phm Flavonoid thu c t l ch khi quan st cc iu kin khc nhau
Vt
(1)

Rf
(2)

Mu sc nh
sng thng
(3)

nh hp ph cc
i ( Max)
(4)

u tin khng xut

hin, sau 1 thi gian
b oxy ho thnh mu
Vng nu
u tin khng xut
hin, sau 1 thi gian
b oxy ho thnh mu
Tm hng

nh 214,4; 274

nh 223,9; 321,7

nh 223,9; 225,1;
276,5
nh 209; 326,3; vai
274,3
nh 220,4; 276,4

0,18

0,23

0,27

Nu nht

0,36

Vng nu to

0,39

Vng nu

6a

0,42

Nu nht to

6b*

0,47

Nu nht

0,56

u tin khng xut

hin, sau 1 thi gian
b oxy ho thnh mu

0,64

Vng chanh to

0,70

Nu en

10

0,74

Vng nht m

NH3

(5)
Vng
chanh 3+

iu kin pht hin

AST khi c phun cc thuc th
UV = 365 nm khi c phun cc thuc th
Vanillin 1% /
Diazo
FeCl3 5%
UV = 356 nm
AlCl3
Willson
NaOH
HCl c (6)
(7)
(8)
(10)
(12)
(13)
(11)
Vng

Vng cam
2+

Xanh en
4+

Nu

Nu +

Pq vng

Vng nu 2
+

Vng
chanh 3+

son 2+

Vng cam
2+

Xanh en
3+

Nu

Nu 2+

Pq vng

Vng cam
3+

Tm hng
4+
Vng cam
4+
Nu en
4+
Nu 4+

son 3+

Vng cam
4+

Xanh en
4+

Nu

Nu 3+

Pq vng

son 4+

Vng cam
4+

Xanh en
4+

Nu to

Nu 3+

Pq vng

son 2+

Vng cam
2+

Xanh en
3+

Nu to

Nu en 3+

Pq vng

Vng cam
3+
Vng cam
2+
Nu 2+

son +

Vng cam
4+

Nu t0

Nu en 2+

Nu +

Pq xanh l

Nu 3+

Vng cam +

Nu

Nu dn

Nu

vng

nh 214,8; 321,0

Vng
chanh 4+

son 2+

Vng cam
4+

Xanh en
2+
Xanh en
3+

Nu to

Nu +

Nu +

Pq xanh l

nh 235,4; 323,4

Vng
chanh 2+
Vng nht
+
Vng nht
+

Xanh en
2+

Nu to

Pq xanh lam

Nu +

Pq vng

Vng +

Nu

Nu

Pq vng
xanh
-

nh 212,9; 273,5;
323,9
nh 214; 300,6

Vng chanh

Ghi ch:
Du +: ch phn ng dng tnh
Du -: ch phn ng m tnh
Con s: ch mc m nht ca mu vt sc k

Pq: Pht quang

*: Vt 6b ch c nhm ch Shan

44

Cc kt qu thu c cho thy:

+S vt tch ra trn bn mng sc k trong cc cp ch phm ti v kh ca tng
ging ch l ging nhau. Cn c trn sc k c th nhn xt: qu trnh x l t mu
ti thnh mu kh hu nh khng nh hng n thnh phn nh tnh ca cc ch
phm Flavonoit
+Sc k ca tt c cc ch phm Flavonoid ch u ging nhau: s vt tch ra trn
sc k cc mu u = 10 vt, vi cc gi tr Rf tng ng; tuy nhin ging ch
Shan Nm Ngt, Shan Cht Tin, Shan Tuyt c 1 vt khc hn so vi cc ging ch
cn li vt 6b (xem hnh 2 v bng 6)
+Quan st sc k khi x l bn mng trong nhng iu kin c trng ca nhm
cht Flavonoid nhn thy:
-Cc vt 1,2,3,4,5,6a,7 cho phn ng c trng ca nhm cht Flavonoid: c phn ng
dng tnh rt r vi thuc th FeCl3; c bit l pht quang vng vi thuc th
Willson khi quan st UV = 365 nm nn c th nhn thy y l nhng Flavonoid c
cu to orthodiphenol, c nhm cacbonyl >C = O v tr C4 v nhm hydroxyl (OH)
t do C5 ; hn na vt 2,3,4,5,6a, 7 cho phn ng (+) vi thuc th Vanillin v thuc
th Diazo, chng t 6 vt ny l catechin hoc l cc dn xut ca Catechin, vt s 1
c th l mt Flavononol. Vt 8,9,10 m nht; khng xc nh r, ch chim mt t l
nh trong tp hp cc cht Flavonoid ch.
-Ngoi 10 vt xut hin trn sc k Flavonoid ca tt c 8 ging ch, 3 ging ch
Shan cn xut hin mt vt vi Rf = 0,47 v cho phn ng dng tnh vi thuc th
FeCl3.
+Tip chng ti tin hnh chy sc k iu ch tch cc vt v o ph t ngoi,
kt qu ct (4) ca bng 6 cho thy xc nh c ph t ngoi c trng ca
tng vt. Vt s 1,3,4 c ph t ngoi nm trong gii II; cn vt s 2,5,6a,6b,7,9 c
ph t ngoi nm trong c 2 gii hp ph ca nhm cht Flavonoid: gii I (hp ph
cc i bc sng > 290 nm) v gii II (hp ph cc i bc sng 220 -280 nm).
Nh vy, hu ht cc cht tch ra trn sc k u c hp ph trong gii
hp th c trng ca Flavonoid (tr nhng vt c t l nh qu khng tin hnh o
ph t ngoi c).
Kt hp ph t ngoi v cc c im sc k bn mng thu c (phun cc thuc

th Diazo, FeCl3, Vanillin, Willson, AlCl3) c th nhn xt: ch phm Flavonoid l

ch bao gm nhiu cht Flavonoid khc nhau, c cu to orthodiphenol, c nhm
cacbonyl >C = O v tr C4 (c th l cc Flavonol hoc Flavon), c bit c nhiu
dn xut Catechin y l nhng c im ho hc rt c gi tr v cn c i vi
mt hot cht t nhin c bn cht polyphenol.

45

4.2 NGHIN CU TC DNG SINH HC

T cc kt qu nghin cu v phn ha hc ca cc mu l ch
chng ti nhn thy thnh phn Polyphenol c trong l ch l cc hp cht
Flavonoid (bao gm: catechin, cc dn xut ca catechin v mt s
Flavonoid khc), chnh v th trong phn nghin cu v hot tnh sinh hc
ny chng ti s dng cc ch phm Flavonoid thu c lm mu
nghin cu.
4.2.1 Nghin cu tc dng chng oxy ho (Antioydant) ca cc ch phm
Flavonoid chit xut t cc ging ch khc nhau i vi nhm mu O ca
ngi.
Mt trong nhng c ch sinh ha hc ng vai tr ch cht quyt nh
hot tnh sinh dc hc ca nhm hp cht Polyphenol l tc dng chng oxy
ha (antioxydant) bi nhiu Polyphenol chnh l nhng cht chng oxy ha
hu hiu gip c th ng vt v ngi phng chng nhiu bnh. Tnh cht
chng oxy ha ca Polyphenol ng vai tr rt quan trng trong vic ngn chn
s hy hoi c th, pht trin v pht sinh nhiu loi bnh him ngho nh
bnh tim mch, ung th, thn kinh, thoi ha, lo ha, ri lon chuyn ha c
bn...Chnh v th chng ti dng ch tiu chng oxy ha lm Test sinh hc
phn tch, sng lc la chn cc mu nghin cu c cht lng Polyphenol tt.
sng lc la chn nhng mu ch c thnh phn Polyphenol tt
chng ti la chn cc ch phm Flavonoid thnh phn Polyphenol ch yu
ca ch tin hnh th nghim th hot tnh chng oxy ha. Trong th
nghim ny, chng ti bc u nh gi tc dng chng oxy ha ca cc ch
phm Flavonoid thng qua hiu lc km hm phn ng oxy ha indigocarmin
bi enzym peroxydaza mu ngi. Hot tnh enzym peroxydaza c tnh theo
cng mu ca phn ng oxy indigocarmin. Hot tnh enzym peroxydaza
mu khng c Flavonoid (mu i chng) l 100%, khi c Flavonoid hot tnh
enzym s thay i. Gi tr IC50 (nng c ch 50% hot tnh enzym) c
tnh bng phn mm x l s liu Raw data. Th nghim c tin hnh vi cc
nng Flavonoid t 0,5g/ml - 128g/ml v trn nhm mu O ca ngi
l nhm mu chim t l cao ca ngi (% t l gia cc nhm mu nh sau:
nhm mu O 45 - 50% , nhm mu A 35 38 %, nhm mu B 12 %,
nhm mu AB 5 %).
Cc kt qu c trnh by bng 7 v biu 5:

46

Bng 7: Tng quan gia nng cc cht th (cc cht Flavonoit thu c t cc ging ch)
vi % c ch hot ng enzym peroxydaza
Nng
cht th
(g/ml)
128

F1

F2

F3

F4

F5

F6

F7

100

92

100

98

100

83

100

94

100

32

46

68

62

59

71

54

67

58

28

25

26

19

24

32

17

10

16

14

17

0,5

12

39,11
2,00

21,95
0,5

24,00
2,2

26,60
0,58

IC50
(g/ml)

21,28
0,38

27,64
1,53

% c ch hot ng ca enzym
F8
F9
F10

F11

F12

F13

F14

F15

F16

100

100

100

100

93

100

100

48

88

46

43

52

52

76

46

27

33

26

31

20

21

12

25

24

10

12

16

17

18

19,76
1,44

39,11
2,14

23,84
0,84

Ghi ch:
F1: Ch phm Flavonoid ca ging Thanh Tm, l ti
F2: Ch phm Flavonoid ca ging Bt Tin, l ti
F3: Ch phm Flavonoid ca ging Kim Tuyn, l ti
F4: Ch phm Flavonoid ca ging Shan Nm Ngt, l ti
F5: Ch phm Flavonoid ca ging Shan Cht Tin, l ti
F6: Ch phm Flavonoid ca ging Shan Tuyt, l ti
F7: Ch phm Flavonoid ca ging Trung Du l xanh, l ti
F8: Ch phm Flavonoid ca ging ch lai LDP1, l ti

25,81
3,06

42,16
0,34

17,29
1,55

39,11
0,34

43,78
0,34

30,45
2,53

30,80
3,02

F9: Ch phm Flavonoid ca ging Thanh Tm, l kh

F10: Ch phm Flavonoid ca ging Bt Tin, l kh
F11: Ch phm Flavonoid ca ging Kim Tuyn, l kh
F12: Ch phm Flavonoid ca ging Shan Nm Ngt, l kh
F13: Ch phm Flavonoid ca ging Shan Cht Tin, l kh
F14: Ch phm Flavonoid ca ging Shan Tuyt, l kh
F15: Ch phm Flavonoid ca ging Trung Du l xanh, l kh
F16: Ch phm Flavonoid ca ging ch lai LDP1, l kh

47

Ga tr
IC50 (g/ml)

Mu ti
Mu kh

45
40
35
30
25
20
15
10
5
0
1

Biu 5: So snh gi tr IC50 ca cc ch phm Flavonoid thu c t cc mu ch

Ghi ch:
1: Ch phm Flavonoid ca ging Thanh Tm
2: Ch phm Flavonoid ca ging Bt Tin
3: Ch phm Flavonoid ca ging Kim Tuyn
4: Ch phm Flavonoid ca ging Shan Nm Ngt
5: Ch phm Flavonoid ca ging Shan Cht Tin
6: Ch phm Flavonoid ca ging Shan Tuyt
7: Ch phm Flavonoid ca ging Trung Du l xanh
8: Ch phm Flavonoid ca ging ch lai LDP1

*Kt lun:
-Cc kt qu thu c bc u cho thy tt c nhng ch phm thu c u c hot tnh
chng oxy ho rt tt: nng 128 g/ml hot tnh chng oxy ho (= % c ch hot
ng ca enzym) ca cc ch phm l t 83 % 100 %, nh vy cc mu nghin cu
u th hin hot tnh chng oxy ho gn nh l trit .
-Phn ln cc ch phm Flavonoid chit xut t l ti th hin hot tnh chng oxy ha
tt hn cc ch phm Flavonoid chit xut t l kh, tuy nhin mu F10 (ch Bt Tin, l
kh) v F15 (ch TDLX, l kh) li l nhng mu c nng IC50 tt nht vi gi tr tng
ng l 17,29 g/ml v 19,76 g/ml.
-So snh gi tr IC50 ca tng vng ch nghin cu nhn thy:
.Trong vng ch Bo Lc - Lm ng: gi tr IC50 gim dn theo th t Bt Tin <
Kim Tuyn < Thanh Tm.

48

.Trong vng ch Shan: gi tr IC50 gim dn theo th t Shan Cht Tin < Shan
Tuyt< Shan Nm Ngt..
.Trong vng ch Tn Cng Thi Nguyn: gi tr IC50 gim dn theo th t
TDLX < LDP1.

4.2.2 Nghin cu tc dng khng khun ca ch phm Flavonoid chit xut t

cc ging ch khc nhau.
Trong dn gian, l ch thng c s dng cha mt s bnh vim nhim nh
gh l, mn nht, vim hng, l...y l nhng bnh lin quan ti cc chng vi khun c
kh nng gy vim nhim bn trong v bn ngoi c th. kim tra hot tnh khng
khun chng ti la chn nhng ch phm Flavonoid chit xut t cc mu l ch kh
(y l nhng ch phm c hm lng Flavonoid cao v hot tnh chng oxy ha tt)
th tc dng khng khun trn 7 chng vi sinh vt kim nh.
+Th nghim c tin hnh trn cc chng vi sinh vt kim nh gy bnh ngi do
ATCC cung cp:
-Trc khun Gram (+): Staphylococcus aureus ATCC 13709; Bacillus subtilis ATCC
6633; Lactobacillus fermentum.
-Trc khun Gram (-): Escherichia coli ATCC 25922; Salmonella enterica; Pseudomonas
aeruginosa ATCC 15442
-Nm: Candida albicans ATCC 10231
+Theo tiu chun sng lc khng sinh thc vt ca hng TIBOTEC (B): ch cng nhn
nhng cht chit xut t thc vt c nng c ch ti thiu MIC 256 g/ml l
khng sinh thc vt. Chng ti cng da vo tiu chun ny sng lc, la chn nhng
mu c hot tnh khng sinh v th nghim xc nh MIC c tin hnh theo phng
php pha long a nng .
Nng ca cc ch phm nghin cu c pha long nh sau: ch phm ban u c
pha trong DMSO c nng l 40 mg/ ml s c pha long thnh cc nng khc
nhau: 256 g/ ml;156 g/ ml; 64 g/ml; 16 g/ ml; 4g/ml; 1g/ml th hot tnh vi
cc chng vi sinh vt kim nh trn. Sau 24h gi nui cy tin hnh c kt qu MIC v
IC50, cc kt qu thu c trnh by bng 8.

49

Bng 8 : Gi tr MIC v IC50 ca cc ch phm Flavonoid chit xut t cc ging ch khc nhau
trn cc chng vi khun kim nh
STT

Tn mu

1
2
3
4
5
6
7
8

Tn chng vi sinh vt kim nh

Staphylococcus aureus
Bacillus
subtilis
IC50
MIC
MBC
IC50 (g/ml)
(g/ml)
(g/ml)
(g/ml)
123,6
256
>256
>256

Candida albicans
IC50 (g/ml)

>256

Pseudomonas
aeruginosa
IC50 (g/ml)
>256

>256

>256

>256

>256

>256

>256

>256

>256

>256

>256

>256

>256

>256

>256

>256

133

256

>256

>256

>256

>256

>256

>256

>256

>256

>256

>256

>256

>256

>256

>256

>256

153,6

256

>256

>256

>256

>256

>256

>256

>256

165,6

256

>256

>256

>256

>256

>256

0,4

0,5

0,5

F9

Lactobacillus
fermentum
IC50 (g/ml)
>256

Salmonella
enterica
IC50 (g/ml)
>256

F10

>256

>256

143,1

256

>256

F11

>256

>256

137,8

256

F12

>256

>256

>256

F13

>256

>256

F14

>256

F15
F16
Amoxicillin

Ghi ch:
F9: Ch phm Flavonoid ca ging Thanh Tm, l kh
F10: Ch phm Flavonoid ca ging Bt Tin, l kh
F11: Ch phm Flavonoid ca ging Kim Tuyn, l kh
F12: Ch phm Flavonoid ca ging Shan Nm Ngt, l kh
F13: Ch phm Flavonoid ca ging Shan Cht Tin, l kh
F14: Ch phm Flavonoid ca ging Shan Tuyt, l kh
F15: Ch phm Flavonoid ca ging Trung Du l xanh, l kh
F16: Ch phm Flavonoid ca ging ch lai LDP1, l kh

50

Escherichia coli
IC50 (g/ml)

>256

Theo kt qu bng 8 cho thy:

-i vi 6 chng vi sinh vt: Bacillus subtilis; Lactobacillus fermentum; Escherichia coli;
Salmonella enterica; Pseudomonas aeruginosa; Candida albicans th tt c cc ch phm
u khng c tc dng c ch trong dy nng nghin cu t 1 g/ml 256 g/ml
-i vi chng Staphylococcus aureus (chng vi sinh vt Gram dng - l chng c
quan tm c bit bi n l nhm t cu khun nguy him c kh nng gy nn qu trnh
vim v sinh m ngi) th kh nng c ch ca tng mu trong dy nng nghin
cu (1 g/ml 256 g/ml) nh sau:
i vi nhm ch Lm ng: Gi tr IC50 (nng c ch 50%) ca F9 (ch phm
t ch Thanh Tm) = 123,6 g/ml > F11 (ch phm t ch Bt Tin) = 137,8 g/ml > F10
(ch phm t ch Bt Tin) = 143,1 g/ml. Gi tr MIC nng c ch ti thiu ca 3
mu trong nhm ch ny u = 256 g/ml.
i vi nhm ch Shan: gi tr IC50 ca F13 (ch phm t ch Shan Cht Tin) =
133 g/ml v gi tr MIC l 256 g/ml. Gi tr IC50 v MIC ca F12 (ch phm t ch
Shan Nm Ngt) v F14 (ch phm t ch Shan Tuyt) u > 256 g/ml
i vi nhm ch vng Tn Cng: Gi tr IC50 ca F15 (ch phm t ch Trung
Du l xanh) = 153,6 g/ml > F16 (ch phm t ch lai LDP1) = 165,6 g/ml. Gi tr MIC
nng c ch ti thiu ca 2 mu u = 256 g/ml.
Gi tr MBC (nng dit khun ti thiu) ca tt c cc mu nghin cu i vi
chng vi khun Staphylococcus aureus khng nm trong gii hn nng nghin cu (
tc >256 g/ ml).
Kt hp gia kt qu phn ha hc vi Test sng lc chng oxy ha + khng khun,
chng ti tip tc la chn mi vng ch nghin cu ra mt ch phm Flavonoid u vit
hn (c hm lng Polyphenol, Flavonoid cao; hot tnh chng oxy ha v khng khun
tt) tin hnh cc nghin cu khc:
-Vng ch Tn Cng - Thi Nguyn: la chn ch phm F15 (Trung Du l xanh)
-Vng ch Bo Lc Lm ng: la chn ch phm F10 (Bt Tin)*
-Vng ch Shan: la chn ch phm F13 (Shan Cht Tin)

(*: Trong nhm ch Bo Lc Lm ng ch Thanh Tm tuy c hot tnh khng khun

tt nht nhng li c hm lng cc cht Polyphenol, hot tnh chng oxy ha km hn
ch Bt Tin, hn na ch Thanh Tm li l ging c nng xut thp nn trong nhm ch
ny chng ti la chn ch Bt Tin lm i din nghin cu tip theo )

51

4.2.3 Nghin cu tc dng gy c t bo trn cc dng t bo ung th ca cc

ch phm Flavonoid chit xut t cc ging ch khc nhau.
Nhng nm gn y, thng c nhiu cng trnh nghin cu v tc dng chng
ung th ca cc hp cht Polyphenol chit xut t ch v c nhiu bng chng cho
thy rng ch xanh vi thnh phn ch yu l cc Polyphenol v c bit l EGCG
(Epigallocatechingallat) c tc dng bo v c th chng li nhiu loi ung th nh phi,
ph qun, tuyn tin lit, tuyn v, gan v bch cu. T l mc ung th tuyn tin lit
Trung Quc mt trong nhng quc gia tiu th ch hng u trn th gii l thp nht th
gii. Cc nghin cu Trung Quc cng cho thy nguy c mc ung th thc qun cng
gim 60% nhng ngi dng t 2- 3 tch nc ch xanh hng ngy.Ch xanh c th
bo v c th khi ung th bng cch lm ngng chu trnh t bo, k c chu trnh nguyn
phn ca t bo ung th. Nhiu nghin cu cp n 2 c ch chnh v tc dng c
ch s pht trin t bo u ca EGCG: (1) tc dng ngn cn chu k phn chia t bo, dng
chu k phn chia t bo cc pha G1, G2; (2) thc y qu trnh apoptosis (s cht t bo
theo chng trnh). Cc nghin cu trn m bnh hc cho thy rng c ch xanh v ch
en u c th km hm s tng sinh ca cc t bo u trong cc m hnh ng vt. EGCG,
EGC v ECG km hm lipoxygenaza u tng mt nhn t thc y pht sin hung
th v pht trin khi u, phn ln c tc dng liu lng thp. Cn theo cc nh khoa
hc M (Neil E.Kay v cs thuc phong th nghim Mayo Clinic) hp cht EGCG
(epigallocatechin 3- gallate) trong ch xanh c kh nng tiu dit cc t bo gy ra bnh
ung th bch cu lympho mn tnh. Kt qu th nghim cho thy, 8 trong s 10 bnh
nhn ung th bch cu nguyn bo lympho mn tnh c t bo bch cu b tiu dit
di tc ng ca EGCG. Mt nghin cu khc ca nh khoa hc trng i hc
Rutgerbcuar (M) cho bit: khi cho polyphenol ca ch en vo mi trng nui cy
ca cc t bo rut kt bnh thng v cc t bo ung thu th thy tt c cc t bo ung th
u b tiu dit trong khi cc t bo bnh thng li khng b nh hng.
Vi mc tiu thm d kh nng ca cc ch phm Flavonoid c hot tnh chng
oxy ha cao tch chit t cc ging ch khc nhau cc vng ch khc nhau, chng ti
tin hnh nhng th nghim:
- Nghin cu tc dng c t bo ( trn cc dng t bo ung th) in vitro ca 3 ch phm
Flavonoid chit xut t 3 ging ch i din cho 3 vng ch ni ting l: Bt Tin, Shan
Cht Tin, Trung Du L Xanh.
- Nghin cu tc dng c chn lc ca ch phm Flavonoid chit xut t ch Trung Du
l xanh (in vitro)
4.2.3.1. Nghin cu tc dng c t bo (trn cc dng t bo ung th) in vitro
+ Th nghim c tin hnh trn 3 ch phm Flavonoid i din cho 3 vng ch:
- F10: ch phm Flavonoid chit xut t ging ch Bt Tin (mu l kh)

52

- F13: ch phm Flavonoid chit xut t ging ch Shan Cht Tin (mu l kh)
- F15: ch phm Flavonoid chit xut t ging ch Trung Du l xanh (mu l kh)
+ Cc dng t bo ung th ngi dung trong th nghim c cung cp bi ATCC
gm: KB (Human epidermic carcinoma), HepG2 (Hepatocellular carcinoma); Lu
(Human lung carcinoma), MCF 7 (Human breast carcinoma), S- 180 (Sarcoma 180)
+ Nng cc ch phm th c dng theo tiu chun sng lc thuc chng ung th c
ngun gc dc liu ca tc gi Teicher 1997. Pha cc ch phm th thnh thang nng
gm 5 nng : 0,5 g/ml; 2 g/ml; 8 g/ml; 32 g/ml; 128 g/ml. S dng phng php
so mu nh gi s tng sinh ca t bo v c s liu trn my Spectrophotometter
Gnios TECAN. Th nghim c lp li 3 ln.
+Cch tnh kt qu: % t bo sng sau khi x l thuc so vi chng trng (t bo
khng c x l bng thuc c gi l % t bo sng, c tnh theo cng thc nh
sau:
% t bo sng = [OD540 ca t bo c x l] x 100%/ [OD540 ca t bo khng c x l cht th]

+Cch tnh gi tr IC50: Gi tr IC50 c phn mm my tnh Table curve.

Cc kt qu thu c trnh by bng 9 v cc biu 6a,b,c,d,e:
Bng 9: Kt qu th c tnh ca cc mu F10, F13, F15 trn cc dng t bo ung th
STT

K hiu
mu

Tn dng t bo
MCF7
IC50 (g/ml)

Lu
IC50 (g/ml)

Hep G2
IC50 (g/ml)

KB
IC50 (g/ml)

S- 180
IC50 (g/ml)

F10

73,45 0,13

69,33 0,24

80,25 0,11

81,48 0,11

72,5 0,15

F13

79,02 0,21

71,16 0,17

80,98 0,09

82,52 0,32

76,4 0,27

F15

62,69 0,42

81,15 0,14

81,48 0,12

82,04 0,25

62,8 0,31

0,31-0,62

0,31-0,62

0,31-0,62

0,62 -1,25

0,31-0,62

Ellipticin

MCF7

Gi tr IC50

Lu
Gi tr IC50

79,02
73,45

81,15

80

82
80
78
76
74
72
70
68
66
64
62

62,69

70
60
50
40
30
20
10
0
1

Biu 6a: Gi tr IC50 ca ch phm F10, F13,

F15 i vi t bo ung th v (MCF7)

71,16
69,33

Biu 6b: Gi tr IC50 ca ch phm F10, F13, F15

i vi t bo ung th phi (Lu)

53

Hep G2

Gi tr IC50

KB
Gi tr IC50

90

81,48

80,98

80,25

80

82,52

81,48

90

82,04

80
70

70
60

60
50

50

40
30

40
30

20
10

20
10
1

Biu 6c: Gi tr IC50 ca ch phm F10, F13,

F15 i vi t bo ung th gan (Hep G2)

Biu 6d: Gi tr IC50 ca ch phm F10, F13, F15

i vi t bo ung th biu m (KB)

S- 180

Gi tr IC50
76,4

80
72,5

Ghi ch:

75

1: ch phm F10 (Flavonoid t ch Bt Tin)

70
62,8

2: ch phm F13 (Flavonoid t ch Shan Cht

Tin)

65
60

3: ch phm F15 (Flavonoid t ch Trung Du

l xanh)

55
50
1

Biu 6e: Gi tr IC50 ca ch phm F10, F13, F15 i vi

t bo ung th (S-180)

Kt hp vic quan st trn knh hin vi v cc kt qu thu c bng 9, cc biu

6a,b,c,d,e cho thy:
+Khi theo di s bin i hnh thi trn knh hin vi thy: ch phm F10, F13, F15 th
hin tc dng c ch cc dng t bo ung th MCF7, Hep G2, Lu, KB, S 180 biu
hin hnh dng t bo b hu hoi v xut hin cc m t bo cht. Khi tng nng
ch phm tc dng gy hu hoi t bo ung th cng th hin r hn.

54

+i vi dng t bo ung th v (MCF7): tc dng gy c 50 % t bo (tc gi tr

IC50) ca ch Trung Du > ch Bt Tin > ch Shan Cht Tin
+i vi dng t bo ung th phi (Lu): tc dng gy c 50 % t bo (tc gi tr
IC50) ca ch Bt Tin > ch Shan Cht Tin > ch Trung Du l xanh
+i vi dng t bo ung th gan (Hep G2) v ung th biu m (KB): tc dng gy
c 50 % t bo (gi tr IC50) ca 3 ging ch nghin cu l tng ng nhau, u
xp x 80 %
+i vi dng t bo ung th bng nc (S - 180): tc dng gy c 50 % t bo (gi
tr IC50) ca ch Trung Du l xanh > Shan Cht Tin > Bt Tin.

Kt lun :
- Ch phm F10, F13, F15 c tc dng ln sinh trng pht trin ca cc t bo ung th,
tc dng ph thuc vo ng nng ooj v i vi tng dng t bo ung th khc
nhau th tc dng c ch cng khc nhau.
- Cckt qu thu c cho thy y l nhng mu c nhiu trin vng v u c gi tr
IC50 < 100g/ml.
4.2.3.2. Nghin cu tc dng c chn lc ca ch phm Flavonoid chit xut t
ch Trung du l xanh (in vitro)
kim tra tnh c chn lc ca ch phm th nghim, chng ti tin hnh
kim tra tc ng ca chng ln lympho bo mu ngoi vi ngi khe mnh.Th
nghim c tin hnh vi ch phm F15 (ch phm th hin tc dng u vit trong th
nghim gy c trn cc dng t bo ung th) v nng th nghim l: 100g/ml v
200g/ml. Cc kt qu thu c trnh by bng 10.
Bng 10: nh hng ca ch phm F15 ln s sinh trng ca t bo lympho
mu ngoi vi ngi ( sau 24 gi nui cy)
Kt qu
Nng ch phm

OD

% so vi C

i chng

1,58 0,26

100

F15 (100 g/ml)

1,49 0,17*

94,30

F15 (200g/ml)

1,46 0,54*

92,40

Ghi ch :* tng ng vi p>0,05

55

Kt qu trn bng 10 cho thy t l t bo sng ca ch phm F15 ti cc nng

th nghim so vi mu i chng khc nhau khng c ngha thng k (P> 0,05). Nh
vy, cc nng th nghim, ch phm F15 khng gy hy hoi trn cc t bo lympho
mu ngoi vi ngi ngi khe mnh, qua th hin tc dng chn lc did vi t bo
ung th v t bo lympho bnh thng.
T tt c cc kt qu nghin cu v ha hc v tc dng sinh hc trn chng ti tip tc
la chn ch phm F15 lm tip mt s nghin cu su hn v:
- y l ging ch rt ph bin vng ch ni ting Tn Cng nn c kh nng cung
cp ngun nguyn liu rt ln.
- L ging ch c tr lng Polyphenol cao, ch yu l Flavonoid hay ni ng hn l
i tng giu catechin
- L ging ch c hot tnh chng oxy ha cao

4.2.4 Nghin cu c tnh cp ca ch phm ch phm Flavonoid chit xut t

ch Trung Du l xanh (F15)
c thm c s cho vic th nghim tc dng sinh hc ca ch phm Flavonoid
chit xut t ch Trung Du l xanh trn cc i tng ng vt v ngi chng ti tin
hnh th c tnh cp ca cc ch phm ny. Th nghim c tin hnh theo nguyn tc
v phng php ca Wallace Hayes.
Cch th nh sau:
-Th nghim thm d cc liu gy c trn ng vt c tin hnh theo nguyn tc v
phng php th ca A. Wallace Hayes.
-ng vt th nghim l chut nht trng chng Swiss, trng lng 20 2g. Mu th l
ch phm Flavonoid chit xut t ch Trung Du l xanh (ch phm F15) c ho tan
trong nc ct, nng ban u ca mu th l 25 mg/1ml.
-Chut c nhn n 15 gi trc khi th nghim, ung nc theo nhu cu. Kim tra cn
nng trc khi th nghim. Chut t cc yu cu v cn nng c a vo th nghim.
-a mu th di dng hn dch theo ng ung. Ly th tch mu th theo qui nh
a thng vo d dy chut bng kim cong u t. Da trn kt qu thm d trong th
nghim s b, tin hnh th nghim chnh thc trn 60 chut, chia thnh 6 nhm gm 1
nhm chng dng nc ct v 5 nhm th theo mc liu d tnh.
-Th nghim c tin hnh trn 50 chut chia thnh 5 nhm, mi nhm gm 10 chut:
Nhm 1 (nhm i chng): cho ung nc ct
Nhm 2: ung 0,15ml mu th /20g trng lng/ngy (tng ng 187,5 mg/kg/ngy)
Nhm 3: ung 0,2 ml mu th /20 g trng lng/ngy (tng ng 250 mg/kg/ngy)

56

Nhm 4: ung 0,35ml mu th /20g trng lng/ngy (tng ng 437,5 mg/kg/ngy)

Nhm 5: ung 0,40ml mu th /20g trng lng/ngy (tng ng 500 mg/kg/ngy)
Nhm 6: ung 0,5ml mu th /20g trng lng/ngy (tng ng 625 mg/kg/ngy)
Theo di chut 7 ngy, k t ngy bt u cho chut ung thuc. So snh vi mu
i chng, ghi nhn cc biu hin lm sng v nhn xt kt qu.
Cc kt qu thu c trnh by bng 11
Bng 11: Kt qu th c tnh cp ca ch phm F15
Cht
th

Nhm chut

Liu dng (ml/20g

chut/ngy)
0,1 ml nc ct

Liu dng (mg/kg

chut/ngy)

S chut
th nghim
10

Kt qu

Sng
Nhm1
(nhm chng)
Nhm 2
0,15
187,5
10
Sng
Nhm 3
0,20
250,0
10
Sng
Nhm 4
0,35
437,5
10
Sng
Nhm 5
0,40
500,0
10
Sng
625,0
10
Sng
Nhm 6
0,50
Nhn xt:
a. Trng lng c th chut
Chut nhm chng v nhm th tng trng lng c th nh nhau trong thi gian theo
di
b. Tiu th thc n v ung ca chut
-Nhm chng: Hot ng v n ung bnh thng
-Cc nhm th: Sau khi ung thuc v trong 7 ngy theo di nhm th, khng nhn thy
c biu hin g khc thng so vi nhm chng.
c. Quan st du hiu ng c
-Khng quan st thy c biu hin ng c cc nhm th, tuy nhin cc chut mc
liu cao trn 500 mg/kg (nhm 5,6) c biu hin hi mt sau khi ung thuc nhng tr li
bnh thng sau 2 gi
-Khng c chut cht trong qu trnh th nghim
Nh vy khi cho chut ung dung dch th F15 trong nc ct vi mc liu t
187,5 n 625mg /kg/ngy, cc kt qu thu c cho thy:
-Khng nhn thy cc chut th nghim c biu hin g khc thng
-Khng nhn thy biu hin ng c cp trn chut th nghim trong thi gian theo
di.Tt c cc chut u n ung, hot ng bnh thng.
-Khng xc nh c liu gy cht 50% ng vt th nghim (LD50) v khng tm
c liu gy cht chut.

4.2.5. Nghin cu tc dng bo v gan ca ch phm F15

Nghin cu tc dng bo v gan ca ch phm F15 thng qua vic kho st kh
nng c ch qu trnh peroxy ho lipid (POL) ca cc ch phm trn ng vt thc

57

nghim gy c bng CCl4; ng thi thm d tc dng iu ho chc nng gan, bo v t

bo gan thng qua s thay i hot tnh cc enzyme AST, ALT trong huyt thanh chut.
Cc enzyme AST (Alanin amino transferase) v ALT (Aspartate amino
transferase) l 2 loi enzym tranrferase trong huyt thanh, xc tc cho qu trnh chuyn
nhm Amin ca axit amin sang nhng axit xetonic. Cc enzyme ny tp trung nhiu t
bo gan v tim. Khi c tn thng hoi t nhng t chc ny th chng vo mu
nhiu hn v lm tng nng trong mu. Trong vim gan do nhim c, x gan AST v
ALT tng rt nhiu. nh lng enzyme trong qu trnh iu tr s gip tin lng mc
ri lon chc nng gan
Th nghim c tin hnh nh sau:
(1) Mu th tc dng bo v gan trong th nghim ny l ch phm F15 . Mu th
c pha thnh dung dch c nng : 10 mg /1 ml. Liu s dng tng dn t 1 5mg
ch phm /1 con chut nng 20g/ ngy. Cho chut ung ch phm trc khi tim CCl4
1ln / ngy trong thi gian 2 ngy (48 gi). Sau tim CCl4 v tip tc cho ung dung
dch th cho n khi kt thc thi gian th nghim (72 gi sau khi tim CCl4).
(2) ng vt c tin hnh th nghim l chut nht trng, c trng lng 20
0,5g.
(3) Tc nhn gy bnh: CCl4 ( tetraclorua cacbon). Cch dng nh sau:
Ho 4ml CCl4 vo 6ml du thc vt c dung dch CCl4 c V = 10ml. Dng hn
hp ny tim cho chut cc l gy bnh (l 2+ 3 ) mt ln duy nht vi liu lng 0,2
ml/ chut.
B tr th nghim: Th nghim tin hnh trong 72 gi, k t khi gy nhim c
CCl4 cho chut.
+ L 1 - i chng (-): (5 chut) ch cho chut ung 0,3 ml NaCl 0,9%/1 ln/ngy
+ L 2 - i chng (+): (5 chut) tim dung dch CCl4 0,2ml/chut v cho ung 0,3 ml
NaCl 0,9% /1 ln/ngy.
+ L 3 - Th nghim: (3 chut x 5 ln lp li = 15 chut). Cho chut ung ch phm F15
vi liu 1,0; 2,5; 5,0 mg ch phm F15/ 1 chut/ ngy, cho ung lin 2 ngy (48 gi) trc
khi tim 0,2ml dung dch CCl4 / mt con chut (mi nng th nghim lp li trn 5 con
chut). Tip tc cho chut ung ch phm F15 n ht 3 ngy (72 gi). Kt thc th
nghim git chut ly mu v ly gan phn tch sinh ho.
Ht 72 gi, git chut nhanh ri ly bnh phm (cho chut nhn n trc 4 ting):

58

- Ly mu, tch huyt thanh nh lng enzym theo phng php ca Reitruan
v Fran Kil trn my phn tch sinh ha t ng Asmes SEAC CH 100, ho cht xt
nghim c hng Span Diagnostic Ltd cung cp.
- Ly gan ti xc nh hm lng DAM. Xc nh hm lng DAM trong gan
nh sau: Ly gan chut va git bc b mng v nghin ng th trong dung dch KCL
1,15% theo t l 1:10 nhit nh hn 100C. Ly 2ml dung dch ng th + 1 m Tris
25mM v 370C trong 60 pht. Sau them 1ml axit tricloacetic 10%; ly tm ly dch
trong cho phn ng vi 1ml axit thiobarbituric 0,25%, t trn ni cch thu n nhit
1000C trong 15 pht v o mu = 532nm. Xc nh hm lng DAM theo phng
php CG.Blagvdorov.
Kt qu th nghim c trnh by trn bng 12 v biu 7:
Bng12: S thay i hm lng DAM trong gan v cc enzym AST, ALT trong
huyt thanh chut b nhim c CCl4 sau 72 gi iu tr bng ch phm F15

TT

L chut
(n = 5)

Liu ch phm
mg/1chut

Hm lng DAM
nM/g Gan

Hm lng AST
(U/ L)

Hm lng ALT
(U/L)

1
2
3

i chng (-)
i chng(+)

0,0
0,0
1,0
2,5
5,0

8,9 0,5
28,3 0,8
20,15 0,07
15,09 0,06
13,9 0,01

14,4 3,39
222,4 22,1
178,16 14,8
148,85 5,6
128,6 17,4

20,2 12,9
182,6 22,4
163,2 11,5
124,71 6,15
122,2 8,13

Ch phm F15

Nhn xt kt qu:
1. Tnh trng chung
Sau 72 gi tt c cc chut u cn sng:
+ L1 - i chng (-): chut kho mnh, n ung bnh thng.
+ L 2 - i chng (+): chut, t hot ng, mt mi, n t.
+ L th nghim (dng F15) :cc chut ung ch phm F15 vi nng 2,5 v 5,0 mg/ chut,
u nhanh nhn, hot ng v n ung tt. cc chut dng liu nh hn chut t ra t
hot ng v n km hn
2. Quan st nhu m gan
Bng mt thng quan st b mt gan ca cc l chut nhn thy:
+ L 1 - i chng (-): b mt gan mn mng, mu nu ti
+ L 2 - i chng (+): b mt ca 1 hoc 2 thu gan ca mi chut c mu bc, c,
khng b hoi t; trong c hai chut c thm vi nt chm nh ang lm m.
+ L dng ch phm F15: cc chut dng liu 2,5 v 5,0 mg/ chut, b mt ca gan mn
mng; quan st bng mt thng khng thy khc bit so vi l i chng (-). chut s
dng liu ch phm thp hn (<1 mg/ chut) trn b mt gan chut xut hin 1 4 nt
chm c m.

59

DAM
AST
250

ALT

200

Ghi ch:

150

1: C (-)
2:C (+)
3: F15 nng 1mg/20g
chut
4: F15 nng 2,5mg/20g
chut
5: F15 nng 5mg/20g
chut

100
50
0
2
3
4
1
5
Biu 7: S thay i hm lng DAM trong gan v AST, ALT huyt thanh chut gy
c gan bng CCl4 sau 72h iu tr bng ch phm F15

(1). Kt qu xt nghim
a. Xc nh hm lng DAM gan chut.
- Cc kt qu thu c cho thy, khi CCl4 gy vim hoi t gan th qu trnh POL cng
tng mnh, lng DAM l i chng dng (+) tng gp gn 3 ln so vi chut khng
gy bnh tc l chut i chng m (-)
- Tc dng chng oxy, ho bo v gan ca ch phm F15 th hin r qua thc nghim:
cc nng nghin cu hm lng DAM gim r rt so vi l i chng dng v gim
tt hn c l nng 5 mg/chut (gim gn 2 ln)
b. Hm lng enzym AST v ALT trong huyt thanh chut
- Hm lng enzym AST v ALT trong huyt thanh ca l chut tim CCl4 nhng khng
iu tr tng [l i chng (+)] rt mnh so vi l chut khng gy bnh [i chng (-)]:
tng gp 15,4 ln i vi AST v 9,03 ln i vi ALT.
- Khi s dng ch phm F15 iu tr th hm lng enzym AST v ALT trong huyt
thanh ti nng 1 5,0 mg tuy vn cao hn so vi l i chng (-) nhng li gim rt
nhiu so vi l i chng (+) v cng gim tt nht l nng 5mg/ con chut: gim
1,72 ln i vi AST v 1,49 ln i vi ALT.
(2). Nhn xt kt qu
- tt c mi liu lng ch phm s dng trong th nghim (t 1,0 5,0 mg/
chut nng 20 gam) u lm thay i hm lng DAM trong gan v enzym AST, ALT
trong huyt thanh.
- Nng ch phm t ra c hiu lc hn c (c hot tnh chng oxy ho tt nht,
th hin qua hm lng DAM trong gan v tc dng n nh chc nng gan tt th hin
qua hm lng enzym trong huyt thanh mu) ca ch phm F15 l 5 mg/ con chut/ngy
hay chnh l nng 250 mg/kg /ngy.

60

4.2.6 Tc dng h lipid huyt ca F15 trn m hnh tng lipid huyt ni
sinh gy bi Triton WR 1339 trn chut nht trng.
-Th nghim c tin hnh theo m hnh gy tng lipid huyt ni sinh bng triton WR
1339 ca Frantz ID v cs, c ci tin. Triton WR 1339 c ch s thanh thi cc
lipoprotein giu triglycerid gy ra s tng lipid huyt cp tnh, thi gian gy tng lipid
huyt ti a l khong 18-20 gi sau khi tim, sau n dn tr v mc ban u.
-Nng ch phm F15 dng trong th nghim tng ng vi liu: 5mg/con chut 20g
= 250mg/kg th trng chut.
-M hnh c tm tt nh sau: Chia chut lm 3 l
+ L C: chng bnh l, ung nc
+ L F15 2: ung F15 liu tng ng 250 mg/kg th trng chut
+ L Feno: ung fenofibrat (thuc ca Php dng iu tr bnh m mu) liu
tng ng 100 mg/kg
Ly mu ui chut nh lng cholesterol v triglycerid huyt thanh [thi im ban u
(to)]. cc l chut ung thuc (cc l th thuc) hoc nc (l chng bnh l) th sau
ngy th nht, ngy th 2 v ngy th 3 sau khi cho chut ung 30 pht, tim tnh mch
ui chut dung dch triton vi liu tng ng 250 mg/kg th trng chut; sau 17 gi
tim triton, tip tc cho chut ung thuc hoc nc v 1 gi sau ung tin hnh ly mu
nh lng cholesterol, triglycerid huyt thanh (thi im ts)
-Phng php nh gi kt qu: Tnh % tng hm lng cholesterol ton phn v
triglycerid huyt thanh ca cng l th nghim trc v sau khi tim triton, so snh mc
tng ca cc l th thuc so vi l chng bnh l, cc kt qu c trnh by bng
13.
Bng 13: Hm lng cholesterol (mg/dl), triglycerid (mg/dl) huyt thanh v % tng
sau 18 gi tim triton so vi trc khi tim.

L th
nghim
C (n=15)
F15
(n = 15)
Feno
(n= 15)

Cholesterol
to

ts

% tng

108,7
3,9
111,0
4,8
104,8
5,0

341,0
12,4
341,1
16,7
245,5
16,3

218,9
16,1
186,21
17
135,4
11,5

Triglycerid
% c
ch

to

ts

% tng

% c
ch

14,93

105,8
5,2
99,0
4,9
102,5
5,8

1081,5
33,7
1058,0
77,4
729,03
58,2

954,4
57,2
849,53
45,2
628,2
60,6

10,98

38,13

P so vi chng bnh l < 0,05

61

34,18

Nhn xt:
-Chut nht trng c ung F15 vi liu tng ng 250 mg/kg/ngy x 3 ngy kt qu
thu c cho thy: F15 c tc dng c ch s tng hm lng hm lng cholesterol v
hm lng triglycerid huyt thanh gy bi triton WR 1339 tng ng l 14,93% v
10,98% so vi l chng bnh l v s gim ny c ngha thng k (P < 0,05).
-Dng thuc i chiu l fenofibrate (liu tng ng 100 mg/kg) th % tng hm lng
cholesterol v triglycerid huyt thanh gy bi triton WR 1339 so vi l chng bnh l
gim r rt, tng ng l 38,13% v 34,18%, s gim ny u c ngha thng k (P <
0,05).
- Nh vy, trong iu kin ca m hnh th nghim ny thy ch phm F15 c tc dng c
ch s tng hm lng cholesterol xp x 25% tc dng ca fenofibrat; cn tc dng c
ch s tng hm lng triglycerid xp x 31% tc dng ca fenofibrat

4.2.7. Nghin cu nh hng ca ch phm Flavonoid chit xut t ch Trung Du

l xanh ln s pht trin ung th trn c i chut gy bi dng t bo S-180
(invivo)
Th nghim c tin hnh trn m hnh chut gy ung th bng cch tim dng t
bo S 180 vi s lng 5 x 106 t bo vo i chut.
iu tr chut man gung th bng cch cho chut ung ch phm F15 hng ngy vi liu
5mg/con chut nng 20g ( tc tng ng vi liu 250 mg/kg/ngy)
nh gi tc dng ca ch phm th thong qua ch tiu: Th tch khi u; t l phn trm hn
ch pht trin khi u ( Ch s GD Growth Delay); trng lng tng i ca u i.
Kt qu thu c trnh by bng 14 v bng 15:
Bng 14: nh hng ca ch phm F15 ln th tch u i chut (tnh bng mm3
Thi gian
sau gy
bnh

L chng
(mm3) n=10

L th nghim vi F14 (n =10)

Th tch u i
(mm3)
68,5 2.1
202,1 8.5
480,8 6.2
876,9 5.8
982,4 6.9

P so vi i
chng
< 0,001
< 0,001
< 0,001
< 0,001
< 0,001

GD(%)

4 ngy
168 5.4
59,22
6 ngy
356,5 17,3
43,30
10 ngy
785,8 10,3
38,81
13 ngy
1084,2 7,8
19,17
15 ngy
1089,3 5,4
9,80
Nhn xt :
- Trong sut thi gian th nghim chut l th nghim ( l ung ch phm F15) vn khe
mnh, nhanh nhn, lng mt n ung bnh thng.
- Chut l i chng, sau ngy th 10 c biu hin x lng, chm chp v xut hin mt
s ri lon tiu ha.

62

Bng 15 : nh hng ca ch phm F15 ln trng lng u i chut

( tnh bng gam, cn vo ngy th 15 sau khi m chut)
L chng

L th nghim vi F15

Trng lng u (g)

Trng lng u (g)

P so vi i
chng

Mc gim trng
lng u (%)

1,53 0,34

1,04 0,42

< 0,05

31,98

Nhn xt:
- Trong sut thi gian th nghim chut l th nghim ( l ung ch phm F15 ) vn
khe mnh, nhanh nhn, lng mt n ung bnh thng.
- Chut l i chng, sau ngy th 15 c biu hin mt mi, b n, chm chp
-Sau khi git chut ly gan v phi quan st cc nt di cn bng mt thng v knh lp
nhn thy: th l nghim ch c 1 -2 nt di cn, cn l i chng c 5 -6 nt di cn.
Cc kt qu bng 14 v 15 cho thy:
Chut nht trng c ung F15 vi liu tng ng 250 mg/kg/ngy x 15 ngy kt qu
thu c cho thy:
- Sau 15 ngy iu tr lin tc bng ch phm F15, th tch khi u i ca chut l th
nghim c gim hn so vi l i chng v c th phn bit c bng mt thng.
- Ch phm F15 c tc dng hn ch s pht trin th tch u giai on sm (4, 6,10, ngy
sau khi gy ung th i thc nghim v cho dung ch phm). n giai on 13 15 ngy,
tc dng ny t thay i v gn nh ngang nhau c hai l th nghim v khi u pht
trin qu ln.
- Trng lng u i ngy th 15 sau khi m chut l th nghim so vi l i chng
gim 32,35 % v s gim ny c ngha thng k vi P < 0,05.
-Ch phm F15 c tc dng hn ch di cn ca t bo u S-180 t i ln gan v phi.
Tuy y mi ch l mt s nhng kt qu kho nghim ban u, nhng cng cho
thy ch phm F15 rt c trin vng trong vic chng ung th v chng di cn nn cn
c nhng nghin cu su hn.

63

T tt c cc kt qu nghin cu ca ti, chng ti nhn thy cy ch Vit

Nam (Camellia sinensis) c y cc c im v thnh phn ha hc v tc dng
sinh dc hc gi tr ging nh cy ch ca nhiu nc trn th gii c nghin
cu v gii thiu trong cc cng trnh c cng b:
-V thnh phn ha hc: cy ch Vit Nam c cha lng ln hp cht
Polyphenol c bit l rt giu hp cht Flavonoid (bao gm ch yu l catechin v
cc dn xut ca catechin, ngoi ra cn c mt lng nh cc cht Flavonoid khc).
-V tc dng sinh dc hc: cy ch Vit Nam c kh nng chng oxy ha
cao, c tc dng khng khun, c tc dng gy c chn lc trn cc dng t bo ung
th, c kh nng bo v tim mch thng qua tc dng h cholesterol v lipid mu v
bc u nhn thy c tc dng km hm ung th trn ng vt thc nghim.

64

Phn 5: KT LUN V KIN NGH

T cc kt qu nghin cu ca ti cho php rt ra mt s kt
lun v kin ngh sau:

I. KT LUN
1.Hm lng Polyphenol tng s, Tanin tng s, Flavonoid tng s ca cc mu
ch u chim mt t l tng i cao:
-Hm lng Polyphenol: mu ti chim t 5,41% 11,15 %; mu kh chim
t 13,98% - 24,40%; hm lng Polyphenol cao nht ch Shan Nm Ngt kh
(= 24,40%) v thp nht l ch Shan Tuyt (=15,21%) ch lai LDP1 (= 15,60%).
- Hm lng Tanin: mu ti chim t 6,74 8,10 %; mu kh chim t
14,86% - 18,52%; hm lng Tanin cao nht ch Trung Du l xanh kh v thp
nht ch Bt Tin kh.
+Hm lng Flavonoid: mu ti hm lng Flavonoid chim t 1,82%
3,04 %; mu kh hm lng Flavonoid chim t 2,51% - 5,77%; hm lng
Flavonoid cao nht l ch Trung du l xanh kh (= 5,77 %) v thp nht l
ch Shan Tuyt kh (=2,51%).
2. Sc k Flavonoid ca 8 ging ch ging nhau l u xut hin 10 vt vi cc
gi tr Rf, ph t ngoi v cc c im sc k tng ng nhau. c im
chung ca thnh phn Flavonoid l ch nh sau: c cu to orthodiphenol, c
nhm cacbonyl >C = O v tr C4 v c nhiu dn xut Catechin
-Tuy nhin, 3 ging ch Shan cn xut hin thm 1 vt (vt 6b) khc vi cc
ging ch cn li.
3. Tt c cc ging ch nghin cu u c hot tnh chng oxy ho rt tt: nng
128 g/ml hot tnh chng oxy ho ca cc ch phm Flavonoid chit xut t
ch l 83 % 100 %. Ch Bt Tin, l kh (F10 ) v ch lai LDP1, l kh (F16 )
l 2 mu tt nht vi gi tr IC50 tng ng l 17,29 g/ml v 19,76 g/ml.
4.Trong 7 chng vi sinh vt kim nh nghin cu, th cc ch phm Flavonoid ca
l ch ch c tc dng trn chng Staphylococcus aureus.
-Gi tr IC50 ca cc mu gim dn theo th t sau: Thanh Tm (F9 ) > Shan Cht
Tin (F13) > Kim Tuyn (F11) > Bt Tin (F10) > Trung Du l xanh (F15) > LDP1
(F16) > Shan Nm Ngt (F12) = Shan Tuyt F14 .

65

-Ch Thanh Tm (F9) c tc dng c ch tt nht vi gi tr IC50 = 123,6 g/ml v

MIC = 256 g/ml.
-Ch Shan Cht Tin v Shan Tuyt c tc dng c ch km nht vi gi tr IC50
v MIC > 256 g/ml
5. Ch phm F10, F13, F15 c tc dng c ch s pht trin ca cc dng t bo ung
th nui cy MCF7, Hep G2, Lu, KB, S -180; tc dng ph thuc vo tng
nng v i vi tng dng t bo ung th khc nhau th tc dng c ch cng
khc nhau. C 3 ch phm nghin cu ny u c gi tr IC50 < 100 g/ml.
Ch phm F15 th hin tc dng gy c t bo mt cch chn lc i vi t bo
ung th v t bo lympho bnh thng.
6. Ch phm F15 (Flavonoid chit xut t ch Trung Du l xanh) c tnh an ton
cao; trong mc liu t 187,5 - 625mg /kg/ngy chut vn kho mnh bnh thng,
khng nhn thy du hiu ng c.
7. Ch phm F15 c kh nng phng v chng tnh trng nhim c gan trn chut
b nhim c CCl4 (in vivo) nhng nng thch hp l 5,0 mg /con chut
tng ng 250 mg/kg.
8. nng 250 mg/kg/ngy ch phm F15 c tc dng c ch s tng hm lng
hm lng cholesterol v hm lng triglycerid huyt thanh chut gy bi triton
WR 1339 tng ng l 14,93% v 10,98% .
9. Ch phm F15 c tc dng c ch s pht trin ca ung th c i gy ra bi t
bo ung th S -180 ti nng 250mg/kg/ngy.

II. KIN NGH

Trong cc kt qu thu c ca ti cho thy ging ch Trung du l xanh
ca vng ch Tn Cng Thi Nguyn l ging ch c nhiu tc dng u vit
nht nn rt c trin vng ng dng vo thc tin. Chnh v vy, chng ti knh
ngh Lin Hip Hi v cc cp c thm quyn tip tc to iu kin v h tr kinh
ph Trung Tm c th tip tc thc hin cc nghin cu su hn v ch Trung
Du l xanh hng ti hnh thnh sn phm thuc hoc thc phm chc nng c tc
dng bo v sc khe cng ng.

66

TI LIU THAM KHO

I. TING VIT
1.Djemukhatze K.M. (1981),Cy ch Min Bc Vit Nam, NXB Nng nghip,
H Ni
2. Dc in Vit Nam (1977), Nh xut bn Y Hc
3. Ngc Qu, Nguyn Kim Phong (1997), Cy ch Vit Nam, NXB Nng
nghip, H Ni
4.on Hng Tin, Vn Ngc (1998), Tuyn tp cc cng trnh nghin cu v
Ch, Nh xut bn Nng nghip, H Ni
5. Tt Li (2000) Nhng cy thuc v v thuc Vit Nam, Nh xut bn KH
& KT, H ni
6. Trung m (2006), Phng php nghin cu tc dng dc l ca thuc
t tho dc, Nh xut bn KH & KT, H ni
7. Th Gm (2002), Nghin cu mt s c im ha hc v tc dng sinh hc
ca cc cht thc vt th sinh trong cy ch camellia sinensis, Lun n tt nghip
Thc s- Khoa sinh hc, i hc KHTN.
8. Hng dn th c tnh cp v c tnh di ngy ca thuc B Y T ban hnh
thng 12/ 2006.
9.L.Z. Rusixky, Hong Hu Nh, Nguyn Bc Vn (dch) (1972) phng php
ton hc x l kt thc nghim Nh xut bn KH & KT, H Ni.
10. Nguyn Vn n, Nguyn Vit Tu (1985), Phng php nghin cu ho
hc cy thuc, Nh xut bn Y hc.
11. Nguyn Quang Trng (1995), Cc phng php xc nh v mt s xu
hng nghin cu thuc gn vi qu trnh peroxy ha, Tp ch dc hc,3, tr. 23
-26.
12.V Thi Th, on Hng Tin v Cs (2001) Cc hp cht ha hc c trong ch
v mt s phng php phn tch thng dng trong sn xut ch Vit Nam ,
Nh xut bn Nng Nghip
13.Vin nghin cu Ch (1994), Kt qu nghin cu khoa hc v trin khai cng
ngh v Ch (1989 - 1993)

67

II. TING ANH

14.Beverly A. Teicher (1997) Anticancer drug development guide: Preclinical
screening, clinical trials, and approval. Humana Press, Totowa, New Jersey.
15.Bors. W. Heller. W, Michel. C, Sarau M. (1990) "Antioxydant in therapy and
priventive medicine"; Radical chemistry of Flavonoit antioxydants. Plemum press,
New York, pp 165 - 170.
16.Blagodorov et al (1987), Pharm Chemi. J. N0, pp 292 - 299
17.Byung Zun Ahn. (1995), Proceeding of UNESCO regione seminar on the
chemistry, pharmacology and clinical use of Flavonoit compounds; Polyoxyfenate
Flavones; synthesis, cytotoxicities and Autitumor activity against ICR Maice
carrying S-180 cell". Korea Oct. 11-85, pp 107 113
18.Chen, C. W and Ho, C. T. (1995), Antioxidant properties of polyphenols
extracted from green tea and black tea, J. Food Lip. 2, pp 35 46
19.(Russian) Epmakov A.U.u gp (1972) Phytobiochemical methodl, Leningrat
Publishing House, pp 117 123, 336 374.
20.Gressman (1975), The chemistry of flavonoid compounds, Academic press,
London.
21.Hara, Y. (1992), Effects of a polyphenols on cardiovascular diseases, Prev.
Med, 21, pp 333
22.Harborn J. B (1964), Biochermistry of phenolic compounds, Academic press,
London and New york
23.Harborn J. B (1986) Nature, distribution and function of plant flavonoids, in
Plant Flavonoids in Biology and Medicine: Biochemical, Pharmacological, and
Structure Activity Relationships (Cody V, Middleton E Jr and Harborne J.B eds),
Alan R. Liss, Inc., New York, pp15 -24
24.Hartmann Lucien (1971), Techniques modernes de laboratoire et exploratios
fonctionnel Tom I L expansion scientifique francaise 15 Rue Saint benoit Paris
25.Franz Hadacek, Harald Greger (2000) Testing of Antifungal Natural Products:
Metholdologies, Comparability of Results and Assay Choice, Phytochemical
analysis 11, 137 147.

68

26.Narayana KR., Reddy MS., Chaluvadi MR. (2001) "BioFlavonoits

classification, pharmacological, biochemical effects and therapentie potential". Ind J.
Pharmacol. 33, pp 2- 16.
27.Mosmann T. Rapid (1983), Colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays J Immunol Methods:
pp65, 55-63,
28. La Logia, R.; Supplement I, (1985), Lokale antiphlogististisch Wirkung def
Kamillen Flavon Dtsch. Apoth. Ztg, pp 9-13
29.OECD guidelines for testing of chemical. Acute oral toxicity Fixed dose
procedure OECD 420, (2001)
30.Pan, M. H., Liang, Y. C., Lin Shiau, S. Y., Zhu, N.Q., Ho, C. T. and Lin, J. K.
(2000), Induction of apoptosis by the oolong tea Polyphenol theasinensin a
through cytochrome release and activation of caspase 9 and caspase 3 in
human U 937 cells J. Agric. Food Chem. 48 (12), pp 6337 6346
31. Pual Cos, Louis Maes, Jean Bosco Sindambiwe, Arnold J.Vlietinck, Dirk
Vanden Berghe; Bioassay for antibacterial and antifungal activities; Laboratory
for Microbiology, Parasitology and Hygien, Faculty of
32. Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp,
Belgium, 1 -13, (2005)
33. Swain J, Goldstein J. L (1963), Methods in polyphenol chemistry,
Proceedings of the plant phenolics group symposium, Oxford
34. Wallace Hayes (2000) Principle and methods of toxycology. The Gillette Co.
Boston USA.

69

C QUAN QUN L TI

C QUAN THC HIN TI

LIN HIP CC HI KH & K THUT VN

TRUNG TM NGHIN CU & PHT TRIN CN HA SINH

Gim c

TS. O TH KIM NHUNG

70

nh 12: Cy ch Shan Nm Ngt 26 nm

tui

nh 14: Nng trng ch Bo Lc Lm

ng

nh 13: Cy ch Shan Cht Tin 28 nm tui

nh 15: Nng trng ch Mc Chu

nh 16: Cy ch Shan Nm Ngt 26 nm tui

nh18: Cy ch Bt Tin

nh 17: Cy ch Shan Tuyt- Mc Chu

nh19: Cy ch Thanh Tm O Long