You are on page 1of 122

Luan van thac s

M AU

T thi xa xa, con ngi a biet s dung cac dc


thao e cha benh rat hieu qua. Cho en nay, cac nha
khoa hoc a xac nh c phan ln cau truc hoa hoc
cua cac hp chat co trong cay co co hoat tnh sinh dc
hoc, giai thch c c che cha benh trong dan gian cua
cac hp chat. T o, co the lam giau va tang tac
dung sinh dc cua chung. Khong ch dng lai viec
nghien cu cac hp chat co cau truc t cay co, con
ngi con nghien cu tong hp ra cac dan xuat co hoat
tnh sinh dc hoc manh hn, gop phan vao lnh vc cham
soc sc khoe con ngi ngay mot nang cao.
Tren c s Hesperidin, mot loai flavonoid co trong
nhieu cay thuoc ho cam qut, ac biet vi ham lng rat
ln trong vo qut, mot loai cay co san va trong nhieu
Viet Nam, chung toi thuy phan ra Hesperetin. Hesperetin
cung la mot loai flavonoid ang c nghien cu kha
nhieu tren the gii v tac dung cua no len nhieu loai
benh khac nhau. Vi mong muon gop phan vao viec
nghien cu sau hn cho viec s dung tot cac hp chat
thien nhien Viet Nam, chung toi thc hien e tai nay,
bc au tong hp hp chat trung gian Triacetyl Hesperetin
e tien hanh tong hp mot vai dan xuat cua Hesperetin
sau nay, xet nghiem tnh khang nam, khang khuan cua
cac hp chat tm c. T o lam nen tang e thc
hien nhng bc tong hp cac dan xuat khac tiep theo
trong nhng nghien cu sau hn.

Hoc vien Nguyen Th Bch Tram


Trang 1

Luan van thac s

Phan I:

TONG QUAN

Hoc vien Nguyen Th Bch Tram


Trang 2

Luan van thac s


I. FLAVONOID.
I.1. Phan bo. [1],[3].
Flavonoid la mot nhom hp chat ln thng gap
trong thc vat. Hn mot na rau qua thng dung co
cha flavonoid. Cho en nay a co khoang 4000 chat a
c xac nh cau truc. Ch rieng 2 nhom flavon va
flavonol vi nhom the la OH hoac OCH 3 th theo ly thuyet
co the gap 38.627 chat. Phan ln cac chat flavonoid co
mau vang. Tuy nhien, mot so co mau xanh, tm, o,
mot so khac khong co mau.
Trong thc vat cung co mot so nhom hp chat
khac khong thuoc flavonoid nhng lai co mau vang nh
Carotenoid, Anthranoid, Xanthon.

I.2. Cau truc hoa hoc. [1],[2],[3].


Flavonoid la nhng chat mau thc vat, co cau truc
c ban la 1,3-diphenylpropan ngha la 2 vong benzen A va B
noi nhau qua mot day co 3 carbon hay noi cach khac la
khung c ban gom 2 vong bezen A va B noi vi nhau qua
mot mach 3 carbon ( C 6-C3-C6). Cau truc la mot vong kn
hoac m.
Cach anh so tuy theo day C 3 ong hay h. Neu
day C3 ong, th anh so bat au t d vong vi d
nguyen to oxigen mang so 1 roi anh tiep en vong A,
con vong B anh so phu. Neu day C3 h, th anh so
chnh tren vong B va anh so phu tren vong A.
3
2
2' 3'
5'
8
O
6'
OH
7
4'
1
1' B 4'
4
1 2
B
A
A 1'
3
4
6' 5'
6
5
6
3'
5
2'
O
O
Cau truc flavonoid
Thng cac flavonoid co mang mot hoac nhieu
nhom OH v tr 5 va 7 tren nhan A va v tr 3; 4; 5

Hoc vien Nguyen Th Bch Tram


Trang 3

Luan van thac s


tren nhan B. Cac flavonoid co the hien dien dang t do
hoac dang glycosid. Cac ng thng gap nhat la
ng D- glucose; ke o la D-galactose; L-Rhamnose; LArabinose; D-Xylose; D-Apiose va acid Uronic.
Trong a so trng hp th mach 3 carbon ong vong
vi vong A va tao nen d vong C co cha oxy. D vong C
co the la Dihydroxy pyran, -pyron, Dihydro -pyron.
O

-pyron

Dihydropyran

Dihydro -

pyron
Tai cac vong co nh mot hoac nhieu nhom hydroxy
t do hoac a thay the mot phan, v vay ve ban chat
chung la cac polyphenol co tnh acid.
Cac polyphenol co the phan ng lan nhau qua cac
nhom hydroxy e tao thanh cac phan t phc tap hn
hoac co the lien ket vi cac hp chat khac trong cay
nh cac oza (dang glycozide), hoac protein.

I.3. Phan loai flavonoid.[1],[3].


S phan loai cac flavonoid da vao v tr goc aryl
(vong B) va cac mc o oxy hoa cua mach 3 carbon.
I.3.1. Flavon.
Flavon co cau truc chung 2 vong benzen A va B. vong
B gan vao vong C (pyran) qua day noi C 2.
3'

2'
7

A
6

O
C

6'
3

4'

5'

O Bch Tram
Hoc vien Nguyen Th
Trang 4

Luan van thac s

Cau truc chung cua Flavon


Flavon thng gap nhat la Apigenin va Luteolin.

HO

HO

OH

HO

OH

Apigenin

Luteolin

Hien nay, ngi ta a phat hien hn 130 hp chat


flavon dang t do cung nh glycoside.
I.3.2. Flavanon.
Flavanon khac vi flavon cho khong co noi oi
v tr C2 va C3. Tat ca cac flavanon c phat hien cho
en nay eu co nhom OH vong A hoac B. Trong t
nhien chung thng co ben canh flavon tng ng, co
nhieu trong cac ho Rosaceae, Rutaceae, Leguminosaea,
Asteraceae. Chat tieu bieu la Naringin, Prunnin, Hesperidin.
Gl Rh

OH

2
3
OH

O
Flavonon

O
Naringin

OH

Gl Rh
Gl

OH

Prunnin

OH

Hesperidin

Hoc vien Nguyen Th Bch Tram


Trang 5

OMe

Luan van thac s


Flavanon la nhng chat khong mau nhng khi lam
phan ng vi cyanidin th cho mau ro hn flavon, ngoai ra
flavanon co iem chay thap hn flavon tng ng.
I.3.3. Flavonol.
Khac vi flavon la co them nhom OH C 3. Flavonol
rat pho bien trong t nhien.

O
3

OH
O
Flavonol
Flavonol ket tinh t vang nhat en vang. Hai chat
quan trong nhat thng gap trong cay la Kaemferol,
Quercetin, th en la Myricetin.

OH
HO

OH

HO

OH
OH

OH

OH

Kaempferol
Thong ke tren
1000 loai thuoc thc
vat hat kn th thay
48%
loai
co
Kaempferol, 26% loai
co
Quercetin
va
Myricetin chiem 10%
trong tong so loai
co flavonol.

OH

O
Quercetin

OH
HO

OH
OH

OH

O
Myricetin

I.3.4. Dihydroflavonon.

Hoc vien Nguyen Th Bch Tram


Trang 6

OH

Luan van thac s


Cau truc c ban giong flavonol nhng khong co noi
oi C2-C3. Con goi la 3-hydroxyflavanol hoac flavanol.
Chat pho bien va n gian nhat la 7hydroxydihidroflavonol.

HO

O
7

OH

OH

Dihyroflavonol

7-hydroxydihydroflavonol

I.3.5. Chalcon.
Chalcon co 2 vong A va B noi vi nhau bi 1 mach
h 3 carbon, khong co d vong nh cac flavonoid khac va
so th t carbon bat au t vong B.
Trong t nhien, chalcon eu co nhom OH vong A
hoac B, la nhng chat co mau vang en vang cam, a
tao ra mau vang ti cua nhieu loai hoa. Chalcon co chu
yeu trong mot so hoa cua ho Cuc-Asteraceae. Chalcon
cung co the co trong cac bo phan khac cua cay nh
vo, la, go, qua, re mot so loai nh Acacia chat
chalcon pho bien nhat la Butein. e nhan biet chalcon, co
the dung hi amoniac hoac khoi kiem cua thuoc la, mau
chuyen sang o cam hay o.

4'

5'
A

3'

2'

6' OH
1'

OH

B
6

HO

OH

OH
Chalcon

Butein

I.3.6. Dihydrochalcon.

Hoc vien Nguyen Th Bch Tram


Trang 7

Luan van thac s


La chalcon mat day noi oi , . Loai nay t gap
trong t nhien. Chat tieu bieu la Phloridzin (trong qua
tao), chat nay co ac tnh ngan s hap thu glucose
ruot non va ngan s tai hap thu glucose tieu quan
than. Mot so dihydrochalcon co v ngot gap 2000 lan
ng ma.

HO 4'

Gl

OH

OH

O
Phloridzin

I.3.7. Auron.
La nhom flavonoid co mau vang sang. Khung cua
auron cung co 12 carbon nh cac flavonoid khac, nhng d
vong C ch co 5 canh. So lng cung nh s phan bo
trong cay cung han che. Chat tieu bieu la Sulfuretin (co
trong nhieu loai hoa), Aureusidin.

6
5

1
O

2' 3'
2

3
4

CH 1'

OH

HO

OH
O

4' OH
6'

5'

CH
O

Aureusidin

Hoc vien Nguyen Th Bch Tram


Trang 8

Sulfureti

OH

Luan van thac s


I.3.8. Anthoxyanidin (2-phenylbenzo pyrilium).
Ve
cau
truc,
phan chung
cho
cac
nhom nay khac cac
anthoxyanin la
flavonoid khac cho
HO
O
khong
co
nhom
OH
+
carbonyl C4 va luon
luon co cac nhom oza
OH
gan vao OH. Cac oza
pho bien la glucoza,
OH
galactoza,
arabinoza,
Pelargonidin
fructoza. Sau khi thuy
Pelargonidin mau vang cam hoac hong, Xyanidin mau
hong xn va Delfinidin mau o hoac tm.
OH
OH
HO

O
+

OH

HO

O
+

OH
OH

OH

OH

OH

OH

Xyanidin

Delfinidin

I.3.9. Leucoanthoxyanidin.
La 3,4-diol flavan, khong mau, nhng gap acid bien
thanh anthoxyanidin co mau hong hoac o, rat pho
bien trong cay, nhat la trong vo cay va go.

OH
HO

OH
OH

OH
3,4-diol flavan

Hoc vien Nguyen Th Bch Tram


Trang 9

Luan van thac s


I.3.10. Isoflavon.
La nhom isoflavonoid pho bien nhat va co nhieu
gia tr ve tac dung cha benh, thng gap trong cac
ho Rosaceae, Amaranthaceae, Iridacea, v du: Daidzein co
trong san day, hoac Formonometin co trong cam thao.
O

R 1O

Cau truc Isoflavon


H

OR2

Daidzein:
Formonometin:

R1 = R 2 =
R1 = H, R2 = CH3

I.3.11. Rotenoid.
Ngi ta a biet khoang 15 chat rotenoid, chat
ien hnh nhat la Rotenon co trong cay thuoc ca Derris
elliptica. Tac dung quan trong cua nhom hp chat nay la
diet sau bo, do han che kha nang thu nhan oxy cua
sau bo.

CH3
O

CH2

C
O

O
O
Cau truc chung cua Rotenoid
Rotenon

Hoc vien Nguyen Th Bch Tram


Trang 10

OCH3
OCH3

Luan van thac s


I.3.12. Neoflavonoid.
Thuat ng Neoflavonoid dung e ch mot nhom hp
chat co khung c ban 4-aril croman. Chat au tien phan
lap la calophylolid chiet t hat cay Mu u Calophyllum
inophyllum va mot so loai Calophyllum khac, ho Guttiferae.

7
6

O
12
43

CH3

5
6'

2'

5'

3'
4'
Cau truc Neoflavonoid

Calophylolid

I.3.13. Biflavonoid.
Harborne xem cac Biflavonoid nh cac dime cua
Apigenin. V du nhom Biflavon va Flavanonyl-flavon c goi
la nhom Agathisflavon.

HO

OH

OH
OH

OH

Agathisflavon

Hoc vien Nguyen Th Bch Tram


Trang 11

OH

Luan van thac s


I.4. Vai tro cua Flavonoid.[1],[3].
I.4.1. Vai tro cua Flavonoid trong cay.
Cac nhom phenol cua flavonoid co vai tro trong s
hoa tan cac chat va di chuyen de dang qua cac
mang sinh ly. Mot so flavonoid co tac dung nh mot
so chat chong oxy hoa, bao ve acid Ascorbic (vitamin
C) mot thanh phan quan trong trong te bao thc
vat. Mot so co tac dung c che cac enzym va
cac chat oc cua cay.
Nhieu cong trnh nghien cu ve tac dung c che
va kch thch sinh trng cay cua flavonoid. Nhom
chc hydroxy co vai tro quyet nh ve tac dung
nay, v du : trong cay oi, cac flavonoid co nhom OH
v tr 4 lam tang cng hoat tnh cua enzym trong
khi cac flavonoid co OH 3 va 4 lai co tnh c che.
Flavonoid con tham gia vao s ho hap-quang hp.
Flavonoid ong vai tro tao mau sac hap dan cho
cay, gop phan thuc ay s sinh ton cua cay va
phat trien hoa qua. Nh he thong th giac ac
biet, sau bo rat nhay cam vi cac mau sac cua
cay co. Thc nghiem cho thay, cac loai ong thch
mau xanh va mau vang, cac loai bm thch mau
hong va trang, cac loai ruoi thch mau trang, cac
loai chim sau th lai thch mau o. Trong viec tao
mau, cac flavon, flavonol, auron, chalcon cho mau vang,
trong khi cac anthocyanine cho cac mau hong, o, tm
hoac xanh tham.
Mot so flavonoid khong mau co trong la ong vai
tro nh mot chat bao ve cay. V ang va kho chu
cua flavonoid lam cho ong vat khi an mat cam
giac ngon va khong thch an cac loai cay co nay.
Quercetin vi nong o rat thap a co cam giac
mat ngon, nhng vi lieu 0.2% se gay c che s
bai tiet nc bot.

Hoc vien Nguyen Th Bch Tram


Trang 12

Luan van thac s


I.4.2. Vai tro cua flavonoid trong y hoc.
Powers (1964) a nghien cu hoat tnh khang khuan
cua 24 loai flavonoid tren 10 chung vi khuan. Hau het
cac flavonoid eu c che ho hap va s tai sinh
san nong o 1-2 mol trong moi trng glucoza.
Vi 24 chat kh khong co chat nao khong co tac
dung di 9 trong so 10 vi khuan th.
Trong chng trnh sang loc chat tac dung vi khoi u
a phat hien mot so flavonoid co tac dung vi
mot so dang ung th. Cac dan chat flavonoid co kha
nang dap tat cac goc t do nh HO , ROO. Cac goc
nay sinh ra trong te bao bi nhieu nguyen nhan va
khi sinh ra canh ADN th se gay ra nhng anh hng
nguy hai nh gay bien d, huy hoai te bao, gay ung
th, tang nhanh s lao hoa. Mot so tai lieu gan
ay co noi en tac dung chong ung th cua mot
so chat Leucocyanidin, Leucopelargonidin va tac dung
khang HIV cua mot so dan chat thuoc nhom flavon
nh Chrysin, Acacetin 7- - -D-galactopyranosid.

OH
MeO

MeO

OMe
OH

OR

OH
HO

MeO

OMe
OMe

OH

( Eupatin
: R= H
Eupatoretin
: R=Me )

O
Centaureidin

Flavonoid tao c phc vi cac ion kim loai ma chnh


cac ion kim loai nay la xuc tac cua nhieu phan
ng oxy hoa. Cac flavonoid co 3,5,3,4-hydroxy co
kha nang lien ket tot vi cac ion kim loai o theo
phc
oxychromon,
oxycarbonyl
hoac
3,4orthodioxyphenol.

Hoc vien Nguyen Th Bch Tram


Trang 13

Luan van thac s


Thanh phan cua mang te bao co cac chat lipid de
b peroxyd hoa, tao ra nhng san pham lam roi loan
s trao oi chat cung dan en s huy hoai te bao.
a ra cac chat chong oxy hoa nh flavonoid vao c
the bao ve te bao th co kha nang ngan nga
cac nguy c nh : x va ong mach, tai bien mach,
lao hoa, ton thng do bc xa, thoai hoa gan.
Mot tac dung quan trong cua flavonoid la nang cao
tnh ben cua thanh mach mau. Flavonoid cung vi
acid ascorbic tham gia trong qua trnh hoat ong cua
enzym oxy hoa kh. Flavonoid con c che tac ong
cua hyaluronidase. Enzym nay lam tang tnh tham
cua mao mach, khi enzym nay tha th gay hien
tng xuat huyet di da ma y hoc goi la benh
thieu vitamin P (P-avitaminose). Cac che pham cha
flavonoid chiet t cac loai Citrus nh Cemaflavon,
Circularine, flavonoid t la bac ha nh Daflon, Diosmin,
flavonoid t hoa hoe (Rutin) vi nhieu biet dc khac
nhau a chng minh tac dung lam ben thanh mach,
lam giam tnh don va tnh tham cua mao mach.
Tac dung nay c hp lc cung vi acid Ascorbic.
Tac dung nay co lien quan en cau truc cua
flavonoid. Thc nghiem cho thay cac flavonoid co
nhom OH t do v tr 3, 4 co tac dung tot oi vi
s nang cao tnh ben vng cua thanh mach. Rutin la
chat tieu bieu ve tac dung nay.
Flavonoid c dung trong cac trng hp roi loan
tnh mach, tnh mach b suy yeu, gian tnh mach, tr,
cac benh trong nhan khoa nh xung huyet ket mac,
roi loan tuan hoan vong mac. Cac dan chat
Anthocyanisid co tac dung tai tao te bao vong mac
va c chng minh co tac dung tang th lc vao
ban em.
Tac dung chong oc cua flavonoid the hien lam
giam thng ton gan, bao ve c chc nang gan khi
mot so chat oc c a vao c the suc vat th
nghiem (CCl4, Benzen, Ethanol, CHCl3, Quinin, Norarseol,)
di tac dung cua flavonoid, ngng Ascorbic c on
nh ong thi lng Glycogen trong gan tang. S tch

Hoc vien Nguyen Th Bch Tram


Trang 14

Luan van thac s


luy Glycogen co y ngha quan trong trong viec nang
cao chc nang giai oc gan. Viec s dung mot so
cay co trong ieu tr viem gan, x gan, bao ve te
bao gan rat hieu qua nh : cay Artiso, co biet dc
la Chophytol, cay Silibum marianum co biet dc
Legalon, cay bup dam Hibiscus sabdariff.
Tac dung kch tiet mat the hien cac chat thuoc
nhom flavanon, flavon, flavonol va flavan -3-ol.
Flavonoid the hien tac dung chong co that nhng to
chc c nhan (tui mat, ong dan mat, phe phan,).
Th du : Apigenin co tac dung lam co that phe quan
gay ra bi Histamin, Acethylcholin.
Tren bo may tiet nieu, nhieu flavonoid thuoc nhom
flavon, flavanon, flavonol the hien tac dung thong tieu
ro ret. Th du : Scoparosid trong Sarothammis seoparius,
Lespecapitosid trong cay Lespedeza capitata, Quercitrin
trong la diep ca, Orthosiphol cua cay rau meo
Orthosiphonis.

Tac dung chong loet cua flavanon va chalcon


glycoside cua re cam thao a c ng dung va
cha au da day. Mot so dan chat khac nh
catechin, 3-O-methycatechin cung a c th nghiem
va thay co tac dung chong loet.
Tac dung chong viem cua nhieu flavonoid thuoc
nhom flavon, flavanon, dihydroflavonol, anthocyanin eu
c chng minh bang thc nghiem do cac chat
flavonoid nay c che con ng sinh tong hp
prostagladin.
Ngi ta a s dung Rutin, Citrin, Leucodelphinidin,
Quercetin e ieu tr ban o, viem da, ton thng da
va mang nhay.

Tren he tim mach, nhieu flavonoid thuoc nhom


flavonol, flavan-3-ol, anthocyanin nh Quercetin, Rutin,
Myricetin, hon hp Catechin cua tra co tac dung lam
tang bien o co bop. Th nghiem lam phuc hoi tim

Hoc vien Nguyen Th Bch Tram


Trang 15

Luan van thac s


khi b ngo oc bi CHCl3 , Quinin, Methanol, bnh thng
lai s roi loan nhp.
Cao chiet t cay Bach qua Ginko biloba cha cac
dan chat 3-rutinosid cua Kaempferol, Quercetin va
Isorhammetin a c mot so hang cua Phap bao
che thanh biet dc Ginkogink Tanakan co tac
dung tang tuan hoan mau trong ong mach, tnh
mach, va mao mach. Thuoc dung cho ngi co bieu
hien lao suy, roi loan tr nh, kha nang lam viec
bang tr oc sut kem, mat tap trung t tng, hay
cau gat. Tren he than kinh mot so flavon glycoside
cua hat tao Ziziphus vulgavis var. Spinsus (cha
Spinosin, Swertisin va cac dan chat acyl cua Spinosin)
co tac dung an than ro ret.
Mot so nhom hp chat flavonoid co tac dung
estrogen . Hien tng xay thai cua cu Uc la mot
van e ln a c nghien cu pho bien. Nguyen
nhan chu yeu gay ra hien tng nay la do cu an
mot loai cay co chat Isoflavon la Genistein vi ham
lng 0.7% trong la. Thc nghiem chng minh chat
nay co tac dung estrogen tren chuot nhat. Tac dung
estrogen co the giai thch la do co s giong nhau ve
cau truc hoa hoc gia chung vi chat estrogen
tong hp Dietylstiboestrol.

HO

OH

O
Genistein

OH

OH

HO
Dietylstibostrol

Hoc vien Nguyen Th Bch Tram


Trang 16

Luan van thac s


II. HESPERIDIN.
II.1. Cac loai cay co cha Hesperidin.
Hesperidin thuoc nhom flavonoid, c tm thay cac
loai cay thuoc ho cam qut nh qut, cam, chanh, bi
Hesperidin chiem ham lng ln trong vo cua cay qut.
Mo ta cay qut. [4],[5].
-

Ten khoa hoc


: Citrus deliciosa Tenore,
Citrus nobilis var. deliciosa Swigle, Citrus
reticulata Blanco.
Thuoc ho : Cam qut ( Rutaceae ).

a) Mo ta cay.
Cay qut la mot loai cay nho, la moc so le, n,
mep la co rang ca, vo co mui thm ac biet. Hoa
nho, mau trang, moc n oc ke la. Qua hnh cau hai
au det, mau vang cam hay o, vo mong nhan hay hi
san sui, de boc, mui thm ngon, nhieu hat.
b) Phan bo, thu hai va che bien.
Qut la mot loai cay c trong khap ni trong nc
ta. Nhieu nhat tai cac tnh Nghe An, Ha Tnh, Quang Tr,
Tha Thien, Nam nh, Ha Nam, Bac Can, Thai Nguyen,
Bac Giang, Bac Ninh, Tai Trung Quoc, ngoai cay cung
loai vi qut cua ta, ngi ta con trong mot so loai qut
khac nh : cay ai Hong Cam ( Citrus chachiensis hay Citrus
nobilis var. chachiensis Wong ), cay Phuc Quyet ( Citrus
tangeriana Hort et Tanaka hay Citrus reticulata var. deliciosa H. H.
Hu ) va cay Chau quyet ( Citrus erythrosa Tanaka hay Citrus
reticulata Blanco var. erythrosa H. H. Hu ). Viet Nam, ngoai
cay qut ngot, ta con nhieu loai cay qut khac cha ai xac
nh ten khoa hoc nh : qut giay, qut tau, qut nuom,
c) Thanh phan hoa hoc.

Hoc vien Nguyen Th Bch Tram


Trang 17

Luan van thac s


Trong thanh phan cua cay qut, Hesperidin chiem chu
yeu trong vo qut vi ham lng tren 3.0% tnh theo dc
lieu kho kiet.

II.2. Cau tao cua Hesperidin.[8],[13],[15],[16].


Cac ten thng goi : Hesperidin; Hesperetin-7rhamno-glucoside; Hesperetin-7-rutinoside va (S)-7[[6-0-(6-deoxy-alpha-L-mannop-yranosyl)-beta-Dglucopyranosyl]-2, 3-dihydro-5-hydroxy-2-(3-hydroxy4-methoxyphenyl)-4H-1-benzopyran-4-one.
Cong thc phan t : C28H34O15.
Khoi lng phan t : 610.55.
Cong thc cau tao :
H
OH

O
CH3
H

H
OH

O
HH

OH

OH

CH2

OCH3
O

H
OH

H
H

OH
H

OH

OH

Hnh 1: Cau truc hoa hoc cua Hesperidin.

II.3. Tnh chat vat ly.[6],[8].


Hesperidin co dang bot vo nh hnh, mau trang,
khong mau, khong mui, khong v.
Hesperidin kho tan trong nc (o hoa tan : 1 gam hoa
tan trong 50 lt nc). o tan tang dan trong cac dung moi
sau : Benzen, Chloroform, nc, Axeton, Methanol. Tan tot trong
dung dch kiem.
Tnc = 258 262 0C

Hoc vien Nguyen Th Bch Tram


Trang 18

Luan van thac s

II.4. ng dung cua Hesperidin.


Cha benh trong dan gian [5].
Trong y hoc co truyen, vo qut a tr thanh nhng
v thuoc pho bien trong nhan gian, thng c tieu thu
mot lng rat ln e lam thuoc cha ho, tieu thc, tr
m, rieng hat c dung e cha cac chng thoat v
rat co hieu qua. Hesperidin t vo qut co tac dung lam
ben thanh mach, cha tr, li mat, khang nam, khang
khuan va chong ngng tap tieu cau.
Trong cha tr ung th. [7]
Ung th ai trang la loai ung th ac tnh. Tang an
m, giam lng carbohydrat th cang tang ty le ung th
ai trang.
Ung th ai trang t xay ra nhng ngi an nhieu
rau qua. ieu o co the giai thch nh sau:
- An nhieu rau qua th co kha nang loai tr cac
carcinogen trong tht va m.
- Rau qua cha nhieu chat anticarcinogen, phong
be s phat trien ung th ai trang. Thc te
trong cac th nghiem ong vat, ngi ta thay
cac thanh phan trong rau qua ngan can s
phat sinh ung th.
Hien nay ngi ta biet trong rau qua la giau
flavonoid, chnh chat nay lam giam ty le ung th ai
trang.
Mot cong trnh nghien cu nam 1997 cua Nhat co
lien quan en flavonoid trong ung th ai trang la s dung
2 chat: Diosmin va Hespiridin t qua thuoc ho cam chanh
e theo doi hieu qua phong ung th cua chung.
Diosmin va Hespiridin la hai chat thuoc flavonoid,
ngi ta biet rat ro chung co ban chat antioxydant, tac
dung chong viem va tac dung c che tong hp
prostaglandin. S lam thay oi sinh hp prostaglandin co the
lam bien ieu s sinh ra ung th ai trang ca ngi va
ong vat. Ca hai dung rieng re hoac phoi hp eu lam
giam ty le ung th ai trang chuot.

Hoc vien Nguyen Th Bch Tram


Trang 19

Luan van thac s


Mat khac ngi ta con thay Diosmin va Hesperidin
co tac dung chong bien d, lam bien ieu cac enzym
chuyen hoa thuoc va tac dung chong khi phat ung th
giong nh cac tac nhan ngan can hoa hoc chong ung
th ai trang khac .
Mot cong trnh nghien cu gan ay, ngi ta phat
hien an Diosmin hay Hesperidin hoac phoi hp ca 2 th
phong toa s cam ng enzym ornithin decarboxylase (ODC)
trong mang nhay ruot gia. Enzym nay la mot enzym lam
han che toc o sinh tong hp cac polyamin (nh Diamin,
Spermidin va Spermin ), ma nhng chat nay co lien quan
chat che en toc o tang sinh san trong mot so mo,
s tang nong o ODC c thay sau s bieu hien cua
cac tac nhan carcinogen. Vay co the lam giam ung th
ai trang cua cac chat flavonoid noi tren ngha la qua s
c che ODC dan en tong hp cac polyamin cung giam
trong mau va s sinh san bieu mo ai trang cung t o
b giam.
Hesperidin, Hesperetin giup ngan nga va cha tr
benh tim.[14],[20].
Hesperidin va Hesperetin lam giam nguy c phat trien
cua benh tim mach : x va ong mach, tang huyet ap,
gay ra bi enzym HMG-CoA (3-hydroxy-3-methylglutaryl
CoA). Hesperidin va Hesperetin c che enzym HMG-CoA, la
tac nhan gian tiep tong hp ra acid mevalonic-hp chat
trung gian trong qua trnh tong hp sterol hoac isoprenoid
lam tang lng cholesterol trong mau.
Mot nguyen nhan na gay ra benh tim mach la tac
dung cua enzym ACAT (Acyl CoA cholesterol-O-ocyltransferase),
enzym nay ay manh s ester hoa cua cholesterol trong
mau, lam phat trien cac ai thc bao lipic thanh
mach tao nen chng x va ong mach. Hesperidin va
Hesperetin co tac dung c che hoat tnh ACAT, gop phan
ay lui nguy c mac phai cac benh ve tim mach.
Hesperidin, Hesperetin anh hng len s chuyen
hoa xng.[12], [15],[17],[24].
Hesperidin, Hesperetin co kha nang kch thch s hnh
thanh va c che s thoai hoa xng. Hoat tnh cua

Hoc vien Nguyen Th Bch Tram


Trang 20

Luan van thac s


phosphatase alcalin chuyen hoa tao hnh xng c kch thch
bi s co mat cua Hesperidin, Hesperetin. Vi phat minh
nay, Hesperidin va Hesperetin va la nguon dinh dng,
va la dc pham quan trong cho con ngi trong viec
phat trien xng, phong va chong benh loang xng.
Hesperidin, Hesperetin giup ngan nga va cha tr
benh gan.[20].
Mot so nghien cu mi ay cho thay Hesperidin va
Hesperetin c a vao s dung e phong nga va cha
tr benh gan : viem gan, x gan, ung th gan.
Mot so nghien cu cho thay:[15],[16],[17],[22].
Hesperidin c s dung rong rai trong dc pham,
my pham va thc pham. Hesperidin la chat co hoat tnh
chong oxy hoa, khang viem, hypolipidemic, khang khoi u,
lam ben vng thanh mao mach, ngan nga s chay
mau va ieu chnh huyet ap. Hesperidin co the c che
cac enzym : phospholipase A2, lipoxygennase, HMG-CoA
reductase va cyclo-oxygenase.
Hesperidin lam tang o ben thanh mao mach bi
lam giam o tham cua mao mach. Hesperidin c s
dung e lam giam sot va cac d ng khac do c che
s phong thch histamine. Va hoat tnh chong ung th c
giai thch bi s c che tong hp polyamine.
Ngoai ra Hesperidin con lam tang hoat tnh cua
vitamin-C, ong vai tro nh mot vitamin P, c s dung
trong viec ieu tr benh chay mau li va cac benh do
thieu vitamin-C.
Hesperidin hoa tan trong dung dch nc kiem, khong
hoa tan trong nc va acid nhiet o thng. Khi san
pham nc ngot ong hop cha mot lng nho
Hesperidin, san pham se b uc, lam i giam gia tr cua
san pham, dan en giam gia tr li nhuan cua nha
san xuat. Hoat tnh cua Hesperidin tr nen manh va de
s dung hn khi tron lan vi Alpha-glucosyl Hesperidin vi
mot t le nhat nh tao thanh enzyme-treated Hesperidin.
Hon hp nay hoa tan tot trong dung dch nc, khong
gay ra s lang ong tinh the va cung khong gay uc
san pham ngay ca bao quan trong thi gian dai. Phat

Hoc vien Nguyen Th Bch Tram


Trang 21

Luan van thac s


hien ra enzyme-treated Hesperidin rat tien li trong lnh vc
cha benh, ngan nga hien tng van uc trong cac
san pham nc cam, qut ong hop va gop phan ngan
nga s phai nhat mau sac t nhien.

Hoc vien Nguyen Th Bch Tram


Trang 22

Luan van thac s


III. HESPERETIN.
III.1. Cau tao.[8],[18].
Cac ten thng goi : Hesperetin; 4-Methyl este
xianidanon; 3,5,7-Trihydroxi-4-metoxiflavon; (S)-2,3dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)4-H-1-bezopyran-4-one.
Cong thc phan t : C16H14O6
Khoi lng phan t : 302.27
Cong thc cau tao :

OH
OCH3
HO

OH

Hnh 2 : Cong thc cau tao cua Hesperetin

III.2. Tnh chat vat ly.[6],[8].


Hesperetin co dang tinh the hnh kim hay luc giac,
mau trang, khong mui, khong v.
t tan trong nc, Chloroform, Benzen. Tan trong Methanol,
Axeton va dung dch kiem.
Tnc = 226 228 0C

Hoc vien Nguyen Th Bch Tram


Trang 23

Luan van thac s


III.3. Phan ng ieu che.[19].
Hesperetin c ieu che bang phan ng thuy phan
Hesperidin trong moi trng acid.
H
OH

O
CH3
H

HH

H
OH

OH

O CH2

OH

OCH3
O

H
OH

H
H

OH
H

OH

OH

OH
OCH3

HO
H2SO4
MeOH

C6H12O6

Glucose

C6H12O5

Rhamnose

OH

O
Hesperetin

III.4. ng dung cua Hesperetin.


Hesperetin la chat chong oxy, co kha nang lam
sach hieu qua peroxynitrite. [23]
Peroxynitrit la chat oxy hoa hoat ong do hai tac
nhan oxy hoa superoxide ( O2- ) va nitric oxyde ( NO), co the
oxy hoa cac thanh phan cua te bao nh : cac protein
thiet yeu, no-protein thiols, ADN, protein t trong thap,
mang nhay phospholipid. Peroxynitrite ong gop lam phat
sinh cac benh nguy hiem nh ot qu, benh tim, tam
than, x va ong mach do thieu enzym noi sinh ngan
can (ONOO)- hoat ong.
Hesperetin a c chng minh la co kha nang lam
sach (ONOO)- hieu qua va ang tin cay. Hesperetin cung
chng to kha nang bao ve te bao t tac nhan gay

Hoc vien Nguyen Th Bch Tram


Trang 24

Luan van thac s


hai bi (ONOO)-. Phat hien ra loai thuoc ac hieu lam
sach (ONOO)- la mot bc ngoat ln trong lnh vc cha
benh.
Hesperetin lam giam nguy c ung th. [21].
Hesperetin lam giam nguy c phat trien ung th. Cac
nha khoa hoc a nhan dang vai bioflavonoid t ho cam qut
c che cytochrome enzym P450. Tac hai cua nhng enzym
nay rat nghiem trong, chung kch hoat cac procarcinogen :
khoi thuoc, thuoc tr sau, tr thanh nhng chat sinh
ung th. Cac procarcinogen nay khong the t sinh ra ung th,
nhng co the tr thanh chat sinh ung th sau khi vao c
the con ngi.
Hesperetin co kha nang c che c enzym P450 1B1tranh s chuyen cac tac nhan procarcinogen thanh cac
chat sinh ung th, lam giam nguy c ung th vu, tuyen tien
liet va c che qua trnh giam hocmon sinh duc n do
enzym nay gay ra. Khong ch ngan chan enzym P450 1B1,
Hesperetin con co tac dung manh len cac khoi u. Anh
hng cua Hesperetin len enzym P450 1B1 dan en nhieu
kha nang trong viec cha benh bang hoa hoc tr lieu.
Hesperetin co kha nang cha tr cac benh ve da
hieu qua nh viem da, mun trng ca, [25].
oi vi cac benh do viem tuyen nhn di da, mun
trng ca, Hesperetin a c chng nhan la dc
pham s dung an toan, co hieu qua ieu tr va tham
my cao hn so vi cac phng phap cha tr khac.
Hesperetin tac ong trc tiep len s bai tiet ba nhn
di da va ba nhn. Hesperetin lam giam lng ba
nhn, dau di da va da au hoac toc ma khong gay
tac dung phu nh teratogenecity, suy yeu tuyen yen, c
the suy yeu oi vi nam khi ket hp vi antiandrogens va
acid retinoid. Hn na Hesperetin co the c che s phat
trien cua vi khuan P. acnes, mot loai vi khuan gay ra mun
trng ca.
Hesperidin, Hesperetin anh hng len s chuyen hoa
xng.[trang 20]

Hoc vien Nguyen Th Bch Tram


Trang 25

Luan van thac s


Hesperidin , Hesperetin giup ngan nga va cha tr
benh tim.[trang 20]
Hesperidin, Hesperetin
benh gan.[trang 20]

giup ngan nga va cha tr

IV. TRIACETYL HESPERETIN. [8],[26].


IV.1. Cau tao.
Cong thc phan t : C22H20O9.
Khoi lng phan t : 428.
Cong thc cau tao :

O
C

CH3
OCH3

H3C

O
CH3

O
Hnh 3 : Cong thc cau tao cua Triacetyl
Hesperetin.

IV.2 . Tnh chat vat ly.


Triacetyl Hesperetin co dang bot, vo nh hnh, mau
trang, khong v.
Tan tot trong Aceton, Etylacetat, tan trong Methanol,
Ethanol, dung dch kiem, khong tan trong Ether dau hoa.
Tnc= 139 1410C

Hoc vien Nguyen Th Bch Tram


Trang 26

Luan van thac s


IV.3. ieu che.
O
O

OH

OCH3

OCH3
HO

CH3COONa
(CH3CO)2O

OH

Hesperetin

C CH3

H3 C

C O

O
CH3

C O

Triacetyl Hesperetin

IV.4. ng dung.
Triacetyl Hesperetin la chat trung gian e tong hp
cac dan xuat cua Hesperetin co hoat tnh sinh hoc cao nh
Diosmin, Diosmetin co nhieu ng dung quan trong trong lnh
vc y hoc.

Hoc vien Nguyen Th Bch Tram


Trang 27

Luan van thac s


V. XAC NH HOAT TNH KHANG NAM, KHANG
KHUAN.
V.1. Cac phng phap nh tnh phat hien
tnh khang khuan.
Phng phap nay cho ta thay ro pham vi tac dung
va nhng hieu biet s bo ve kha nang cua loai khang
khuan to ang con giai oan tho .
V.1.1. Phng phap tron thuoc vao moi trng
thach.[9].
Chat chiet c pha loang vi dung dch em co
nong o xac nh. Dung dch nay tron eu vi moi
trng nuoi cay, e nhiet o 48 0C 500C, sau o o ra
tng hop long vi mot lng nhat nh. Khi thach ong
lai ta cay cac chung vi sinh vat a c chuan b t
trc. Dung que cay vo trung lay cac chung vi sinh vat
goc cay len tren mat moi trng thach thanh tng
vach thang. e hop long trong ieu kien thch hp, t 18
en 20 gi cho vi khuan moc len.
Chung ta nhan nh ket qua bang s quan sat vi
khuan moc thanh tng khuan lac, hay khong moc c so
vi mau oi chng.
V.1.2. Phng phap uc lo trong moi trng thach. [9].
Trong hop long, o moi trng thach nong chay,
e ong lai, sau o cay vi khuan eu khap tren mat.
Dung ong khoan lo co kch thc quy nh khoan vao
thach e tao ra mot lo tron. o mau thuoc can nghien
cu vao lo tron o roi e vao tu am 37 0C sau 18 gi.
Phng phap nay cho ket qua ro rang, do chat
khang khuan ngam trc tiep vao moi trng.
V.1.3. Phng phap tham giay. [9].
Giay tham cat thanh hnh tron co ng knh
khoang 33.5 34.0 mm, say vo trung. Cho cac mau giay

Hoc vien Nguyen Th Bch Tram


Trang 28

Luan van thac s


nay tham vao dung dch co cha chat khang khuan, roi
hong kho. at cac giay nay len mat moi trng thach
a gieo cay vi khuan, cat vao tu am 37 0C sau 18 -20 gi,
em ra oc ket qua. Cho thach co chat khang khuan
ngam ra, vi khuan khong phat trien c.
Phng phap nay ch thch hp khi phat hien loai vi
khuan co hoat tnh manh. Ngoai ra con anh hng kha
nhieu en lng vi khuan cho vao moi trng moi th
nghiem khong hang nh.
V.1.4. Phng phap ong tru cua Heatley. [9].
Dung hop long co ng knh 10 cm, ay bang, o
vao khoang 10 ml moi trng thach a un chay, e cho
ac lai. un chay mot bnh moi trng thach khac co
cha 20 ml, ch khi nhiet o moi trng tr ve 42 0C 440C, cho vao 0.2 ml vi khuan a cay vao canh thang moc
tot, trc o 12 gi, lac eu moi trng va vi khuan
roi o ra hop thach noi tren mot lp mong khoang 4
ml. Lp nay se ong lai sau 10 15 phut. at len mat
lp thach nhng vong kim loai khong g, vong nay cao
9.6mm, ng knh ngoai 7.2 mm, ng knh trong 5 mm,
moi vong kim loai co khoi lng khong nhe qua 1.7 gam
e am bao o lun vao thach. o dung dch can
nghien cu vao cac vong noi tren vi nong o va
khoi lng nhat nh, roi nhe nhang e vao tu am 37 0C
khoang 18 gi. Sau thi gian nay ta se thay vi khuan
phat trien thanh mot mau trang uc, eu khap mat
moi trng, con quanh vong kim loai co the co mot
vung trong, vung nay chng to vi khuan khong moc
c.
Phng phap nay rat thng dung cho viec phat
hien tnh khang khuan, co u iem am bao an toan cho
ngi thc hanh ky thuat, t b nhiem trung, ket qua ro
rang.
V.1.5. Phng phap ao ranh cua Fleming (1932). [9].
Phng phap nay dung e kiem nghiem tnh khang
khuan cua cac giong ieu che ra Penixilin. Moi trng
thach nau chay o vao cac hop long, ch cho ong lai,
dung dao nho vo trung cat hai ng song song gan gia

Hoc vien Nguyen Th Bch Tram


Trang 29

Luan van thac s


hop ( hai ng nay cach nhau 0.3 cm ), bo thoi thach
gia i, o dung dch thuoc nh th vao ranh. Cay vi
khuan thanh ng thang goc vi ranh. e vao tu am
370C khoang 18 gi. oc ket qua bang cach o vung vo
khuan t chan ranh tr ra.
V.1.6. Phng phap vien nen ang Van Ng (1956).
[9].
Cac mau thuoc c nghien nat, ep thanh vien
hnh tru, cao 0.8 cm, ng knh 1 2 cm. at nhng vien o
len mot hop moi trng thach, at hop nay va tu lanh
40 C trong 6 gi, e hoat chat khang khuan t dc lieu
khuech tan vao moi trng. Sau o lay ra, m nap hop
long at vao chuong thuy tinh va dung mot dung cu
ac biet phun len tren mat moi trng mot lp vi
khuan, ay nap lai, e vao tu am 370 C trong 12 -18 gi.
oc ket qua bang cach o vung vo khuan quanh vien
nen.
Phng phap nay tranh c phai dung nhieu vong
kim loai nhng phai dung nhng thiet b cong kenh khac.
Cung can phai rat than trong e tranh lay lan khi phai
dung nhng loai vi khuan oc e th nghiem.
V.1.7. Phng phap th nhng chat khang khuan bay
hi. [9].
Phng phap nay c Rundat e xuat lan au tien
khi nghien cu chat khang khuan bay hi t hat u u.
Noi dung cua phng phap nay la : dung hai na
hop long co ng knh bang nhau, o moi trng thach
nong chay vao na hop long pha ay, e cho moi
trng ong lai, cay vi khuan thanh tng ng thang.
Na hop long kia e cac chat tinh dau hay nhng thc
vat co chat bay hi gia dap. Gan khe h bang bang dnh
hoac paraphin, e vao tu am 37 0 C, sau 12 -18 gi oc ket
qua, so sanh vi oi chng. Chat bay hi co tac dung
c che hoac tieu diet vi sinh vat em th.
Phng phap nay cho phep phat hien c nhng
chat bay hi mang tnh khang khuan ma cac phng phap
ke tren khong the phat hien c.

Hoc vien Nguyen Th Bch Tram


Trang 30

Luan van thac s


V.1.8. Phng phap sac ky khang khuan. [9].
Viec phan tch khang khuan to giai oan au
trong qua trnh nghien cu bang phng phap sac ky
giay c nha bac hoc Ichida (80) ap dung trc tien, sau
o a c nhieu ngi cai tien them. Mac du co rat
nhieu tai lieu e cap en viec s dung phng phap
sac ky tren giay nhng cho en nay, van cha co mot
quan iem thong nhat ve he thong hoa cac khang
khuan to bang phng phap nay.
Phng phap tom tat nh sau : Chat em th c
hoa tan trong dung moi thch hp, sau o dung loai dung cu
ac biet e lay chat th, cham nhieu lan vao mot
iem tren giay (loai giay rieng biet e lam sac ky),
ng knh cua vet khong qua 6 8 mm. Cho chay tham
do vi tng he dung moi trong mot bnh thuy tinh kn, a
c bao hoa trc tng khong di ong. Trong sac ky
ngc, t giay c gi pha tren bnh va tha au kia
(au co cham chat th) xuong mang ng tng di
ong ngap ti 2 3 cm, cach iem cham chat th 3 4
cm. Co the dung phng phap sac ky xuoi hoac phng
phap sac ky vong. Sau khi a chay xong phan sac ky,
giay c uoi het dung moi va hong kho. at cac
bang giay tren moi trng thach a cay vi khuan, e
nhiet o 370 C trong 12 gi cho vi khuan phat trien ta se
c vung vo khuan vet sac ky mang hoat tnh khang
khuan.
Phng phap nay co u iem tach biet c chat
khang khuan ra khoi nhieu chat khac. Xac nh c ch
so Rf cua vet khang khuan, cho phep ta i sau nghien
cu thanh phan va cau truc cua chung.
V.1.9. Phng phap hien ai cua Vanden Benergher va
Vlietlinck (1994).[12 ].
Phng phap nay tien hanh th hoat tnh khang vi
sinh vat tren phien vi lng 96 gieng cua cac mau chiet
theo 2 bc :
Bc 1 : Sang loc s bo tm chat chiet co hoat tnh;
Bc 2 : Tm nong o c che toi thieu (MIC) cua
chat co hoat tnh.

Hoc vien Nguyen Th Bch Tram


Trang 31

Luan van thac s


Nam va vi khuan c duy tr trong moi trng dinh
dng. Cac chung kiem nh c hoat hoa trc khi
tien hanh th nghiem trong moi trng dinh dng dch
the (24 gi oi vi vi khuan, 48 gi oi vi nam).
Mau th c hoa tan cac chiet pham trong dung
moi Dimethyl sulfoxid (DMSO) 100%, vi 4 10 thang nong
o c pha loang t dch goc roi nho sang phien vi
lng 96 gieng. Vi sinh vat kiem nh sau khi c hoat
hoa, pha loang bang moi trng dinh dng cho ti nong
o tng ng 0.5 n v McLand (khoang 108 vsv/ml). e
trong tu am 370C trong thi gian 24 gi oi vi vi khuan
va 300C trong thi gian 48 gi oi vi nam. Sau o oc
ket qua va tnh gia tr c che toi thieu (MIC).
Mau tho co MIC 200 g/ml; mau tinh co MIC
50g/ml la co hoat tnh

V.2. Cac loai vi khuan, vi nam s dung e xac


nh hoat tnh.[10],[11].
V.2.1. Vi khuan.
Staphylococcus aureus :
-

La vi khuan Gram (+), khong giap mo, khong


tao bao t.
Sc e khang : nhay cam vi Penicilin,
Authromycin va Ampicilin.
Kha nang gay benh :
+ Sng mu tren da, niem mac,
+ gay ung nhot, ap xe,
+ viem vu bo, cu,
+ gay nhiem trung mau dan en
viem phoi, viem than cap, viem tuyen
sa va viem tuy xng.

Bacillus subtilis :
-

La vi khuan Gram (+), trc khuan.


La vi khuan co li, co mat ng ruot.

Pseudomonas aeruginosa :

Hoc vien Nguyen Th Bch Tram


Trang 32

Luan van thac s


-

La vi khuan Gram (-), co tiem mao, vi khuan sinh


sac to mau xanh.
Kha nang gay benh :
+ Gay benh mu xanh ngi va
ong vat,
+ viem bang quan, viem tai co mu
ngi.

E.coli:
- La vi khuan Gram (-), khong bao t.
- Sc e khang : nhay cam vi Chloramphenicol,
Streptomycine, Sulfamid, Trimrthoprim.
- Kha nang gay benh : E.coli co san trong ruot,
khi c the b suy yeu, sc e khang giam th
E.coli mi gay benh :
+ Gay tieu chay,
+ benh phu thung heo con,
+ viem ruot.
Enterococcus faecalis :
- Enterococcus
la
mot
phu
nhom
cua
Streptococcus, nhom D.
- La vi khuan Gram (+), dang cau, khong bao t.
- Kha nang gay benh : tren da, ng tieu hoa,
da day, ruot va nieu sinh duc.
V.2.2. Nam.
Aspergillus :
-

La loai nam moc co kha nang sinh oc to


gay ung th la Aflatoxin.
ac iem hnh dang : hnh bong mau luc, cuong
sinh bao t.
+ Aspergillus flavus : the bnh, 1 hay 2 tang
tren cung mot bong. Bong hnh phong xa
hay hnh cot long leo, cuong sinh bao t.
+ Aspergillus oryzae : cuong sinh bao t
dai 1 2 mm. The bnh hai lp, khi gia mau hi
nau.
Kha nang gay benh cua Aflatoxin :

Hoc vien Nguyen Th Bch Tram


Trang 33

Luan van thac s


+ gia suc : - Khi trung oc cap tnh :
chet ot ngot.
- Khi trung oc man tnh : con
vat kem an, cham ln, sut
can.
- Khi nhiem oc keo dai : gay
ung th.
+ ngi : gay ung th.
Trichoderma sp :
-

Thuoc dang vi nam, song trong moi trng at.


Hnh dang : co khuan ty khong mau, cuong
mang bao t phan nhanh, cuoi nhanh phat
trien thanh mot khoi tron nh cac bao t
tron mau luc lien ket vi nhau nh chat
nhay. Hnh thc sinh san bang bao t.

Hoc vien Nguyen Th Bch Tram


Trang 34

Luan van thac s

Phan II :

THC NGHIEM

Hoc vien Nguyen Th Bch Tram


Trang 35

Luan van thac s

NOI DUNG NGHIEN CU :


Tren c s Hesperidin, mot flavonoid c chiet tach
t vo qut, chung toi tien hanh tong hp Hesperetin.Theo
cac tai lieu, Hesperidin va Hesperetin la nhng hp chat
co hoat tnh sinh hoc cao, khang nam, khang khuan tot,
la chat chong oxy hoa manh. Ben canh Hesperidin va
Hesperetin, chung toi con nghien cu tong hp Triacetyl
Hesperetin-mot hp chat trung gian, e tien hanh tong
hp cac dan xuat cua Hesperetin sau nay.
Muc tieu nghien cu cua e tai :
-

Tach chiet Hesperidin,


Tong hp Hesperetin,
Tong hp Triacetyl Hesperetin,
Xac nh hoat tnh khang khuan, khang nam
cua Hesperidin, Hesperetin va Triacetyl Hesperetin.

Vi cac noi dung tren, s o nghien cu nh sau :

Hoc vien Nguyen Th Bch Tram


Trang 36

Luan van thac s


La chon
nguyen lieu
(vo qut)
X ly nguyen
lieu
Chiet tach
Hesperidin
cu

S o 1:

S o nghien

Tinh che Hesperidin

Hesperidin
tinh
Khao sat qua trnh tong hp
Hesperetin
Tinh che Hesperetin

Hesperetin
tinh
Khao sat qua trnh
tong hp Triacetyl
Hesperetin
Tinh che Triacetyl
Hesperetin

Triacetyl
Hesperetin
tinh

Xac nh tnh
chat cua san
pham
nh
tnh,
nh
lng

Xac
nh
cau
truc

Hoat
tnh
sinh
hoc

anh gia tnh


chat, kha nang
ng dung

Hoc vien Nguyen Th Bch Tram


Trang 37

Luan van thac s


A. PHNG PHAP NGHIEN CU.
I. LA CHON X LY NGUYEN LIEU.
Trong cac loai thc vat co Viet Nam, cay qut co
ham lng Hesperidin cao, ac biet vo qut vi ham
lng tren 3.0% tnh theo dc lieu kho kiet. V vay chung
toi chon nguyen lieu chnh la vo qut.
Vo qut

Loai tap

Vo qut say kho


(600 C )

Nghien mn

Vo qut
dang
bot
S o 2 : X ly nguyen lieu.

Hoc vien Nguyen Th Bch Tram


Trang 38

Luan van thac s


II. TACH CHIET HESPERIDIN.
II.1. Phng phap va ieu kien chiet
Hesperidin.
Hesperidin la mot hp chat co the tan trong nhieu
dung moi, o tan tang dan trong cac dung moi sau:
+ Benzen
+ Chloroform
+ Nc
+ Aceton
+ Methanol
+ Dung dch NaOH loang
Hesperidin tan tot trong dung dch NaOH loang. Tuy
nhien khi chiet Hesperidin t bot vo qut bang dung dch
NaOH loang bang phng phap ngam dam, ton nhieu
thi gian nhng hieu suat chiet khong cao. Do o chung
toi chon phng an chiet Hesperidin t vo qut bang dung
moi Methanol.
ieu kien tien hanh
Chung toi chon ieu kien toi u e tien hanh
chiet tach Hesperidin nh sau:
Dung cu chiet : soxhlet.
Chiet loai chat beo, chat mau :
+ Dung moi
: Ether dau hoa
+ Nhiet o chiet
: nhiet o soi cua
dung dch.
Chiet Hesperidin :
+ Dung moi
: Methanol.
+ Nhiet o chiet
: nhiet o soi cua
dung dch.

Hoc vien Nguyen Th Bch Tram


Trang 39

Luan van thac s

Hoc vien Nguyen Th Bch Tram


Trang 40

Luan van thac s


Quy trnh chiet :

Ether dau
hoa

Bot vo qut

Chiet chat beo,


chat mau
Dch
chiet
(Thu hoi dung
moi)

Loc
ba
Methano
l

Hesperidin
Chiet
tho Hesperidin

Metano
l

Hoa tan
Loc
Dch chiet
Loc nong

Ca
n

Can
Dch
locMeOH
MeOH lanh

Loc, ra
Loai bt dung
moi
Say
Ket tinh

Nc lanh

Nc qua
Loc, Hesperidi
ra
loc
n tho
S o 3 : Quy
trnh chiet Hesperidin.

II.2. Tinh che Hesperidin.


.
Say
Hesperidin chiet xuat c em tinh che va ket tinh
nhieu lan trong Methanol.

Hoc vien Nguyen Th Bch Tram


Trang 41
Hesperi
din tinh

Luan van thac s

S o 4 : Quy trnh tinh che Hesperidin.

Hoc vien Nguyen Th Bch Tram


Trang 42

Luan van thac s


III. NGHIEN CU IEU KIEN TONG HP
HESPERETIN
III.1. ieu kien va quy trnh tong hp.
ieu kien tien hanh :
Chung toi chon ieu kien toi u e tien hanh qua
trnh thuy phan Hesperidin thanh Hesperetin nh sau :
- Khoi lng nguyen lieu : 5 gam Hesperidin.
- Lng MeOH
: 100 ml.
- Lng H2SO4 96%
: 5 ml.
- Nhiet o phan ng
: tsoi cua dung
dch.

- Thi gian phan ng


: 6 gi.
- Qua trnh thuy phan c khuay lien tuc.

Tien hanh khao sat anh hng cua thi gian, lng
Methanol, acid H2SO4 96% len khoi lng cua san pham
phan ng.

Hoc vien Nguyen Th Bch Tram


Trang 43

Luan van thac s


Chung toi chon quy trnh tong hp Hesperetin nh sau :
Hesperidi
n
Methano
l
H2SO4
96%

Thuy phan

Loc
Ethylacet
at

Dd NaCl
15%
Nc
cat

Hon hp dung
dch

ra

Thu hoi dung


moi
Say

Hespere
tho
S o 5 : Quy trnh tin
thuy
phan Hesperidin thanh
Hesperetin.

Hoc vien Nguyen Th Bch Tram


Trang 44

Luan van thac s


III.2. Tinh che Hesperetin.
Hesperetin tong hp c em tinh che va ket tinh
nhieu lan trong Ethanol.
Hesperetin
tho
Ethano
l
Than hoat
tnh

Hoa tan

Loc nong

Ca
n

Dch loc

Loai bt dung
moi

Ket tinh
Nc lanh

Loc, ra

Nc qua
loc

Say

Hespere
S o 6 : Quy trnh tinh che Hesperetin.
tin tinh

Hoc vien Nguyen Th Bch Tram


Trang 45

Luan van thac s


IV.

NGHIEN CU IEU
TRIACETYL HESPERETIN

KIEN

TONG

HP

IV.1. ieu kien va quy trnh tong hp.


ieu kien tien hanh :
Chung toi chon ieu kien toi u e tien hanh qua
trnh tong hp Hesperetin thanh Triacetyl Hesperetin nh sau :
-

Khoi lng nguyen lieu : 0.2000 gam Hesperetin.


Lng CH3COONa
: 0.075 gam.
Lng (CH3CO)2O
: 3 ml.
Nhiet o phan ng
: tsoi cua dung dch.
Thi gian phan ng
: 1 phut.
Qua trnh phan ng c khuay lien tuc.

Tien hanh khao sat anh hng cua thi gian, lng
(CH3CO)2O, lng CH3COONa len khoi lng cua san pham
Triacetyl Hesperetin.

Hoc vien Nguyen Th Bch Tram


Trang 46

Luan van thac s


Chung toi chon quy trnh tong hp Triacetyl Hesperetin
nh sau :

Hespereti
n
(CH3CO)2
O
CH3COON
a

Ester hoa

Coc nc
a
Nc
cat

Loc, ra

Say

Triacetyl
Hesperetin
tho
S o 7: Quy trnh tong hp Hesperetin thanh Triacetyl
Hesperetin.

Hoc vien Nguyen Th Bch Tram


Trang 47

Luan van thac s


IV.2. Tinh che Triacetyl Hesperetin.
Triacetyl sau khi c tao thanh em tinh che va ket
tinh nhieu lan trong Ethanol.
Triacetyl
Hesperetin tho
Ethano
l

Hoa tan

Loc nong

Ca
n

Dch loc

Loai bt dung
moi
Ket tinh
Nc lanh

Loc, ra

Nc qua
loc

Say

V.

S o 8 : Quy
trnh tinh che Hesperetin.
Triacetyl
Hesperetin
NHAN DANH SANtinh
PHAM.

Hoc vien Nguyen Th Bch Tram


Trang 48

Luan van thac s


San pham c nhan danh bang cac phng phap
phan tch hoa ly hien ai : sac ky lp mong, o nhiet
o nong chay, UV-Vis, IR, HPLC, LC-MS, 1H-NMR, 13C-NMR.

VI. XAC NH HOAT TNH KHANG NAM, KHANG


KHUAN.
Cac san pham tach chiet, tong hp c em th
hoat tnh sinh hoc tai phong sinh hoc thc nghiem-Vien
Hoa hoc cac hp chat thien nhien-Vien KH & CN Viet
Nam.

Hoc vien Nguyen Th Bch Tram


Trang 49

Luan van thac s


B. THC NGHIEM.
I. LA CHON X LY NGUYEN LIEU.
Nguyen lieu s dung chnh la vo quit kho c mua
khu vc ban thuoc bac ng Hai Thng Lan Ong,
em sang e loai tap chat : at, cat; say kho nhiet
o 50-600C. Sau o nghien mn, bao quan kn e tranh
hut am tr lai.

II. TACH CHIET HESPERIDIN.


II.1. Phng phap va ieu kien chiet tach
Hesperidin.
Cac ieu kien tien hanh :
-

Nhiet o chiet : nhiet o soi cua dung


dch.
Dung moi : Ether dau hoa, Methanol.
Chiet lien tuc trong soxhlet.

II.2. Tinh che Hesperidin.


Ket tinh lai trong Methanol.
Can 0.5 gam Hesperidin tho, hoa tan trong 750 ml
Methanol. un soi va khuay nhe bang khuay t cho en
khi tan hoan toan. Loc nong dung dch bang thiet b loc
hut chan khong. Dch qua loc c tiep tuc s dung co
quay chan khong e loai bt dung moi, e lanh dung
dch, Hesperidin se ket tinh lai, loc, ra, say chan khong
800C. em can Hesperidin thu c e tnh hieu suat tinh
che.

Hoc vien Nguyen Th Bch Tram


Trang 50

Luan van thac s


III.

NGHIEN CU
HESPERETIN.

IEU

KIEN

TONG

HP

III.1. Khao sat qua trnh tong hp Hesperidin


thanh Hesperetin.
Lay 5 gam Hesperidin tinh, 100 ml Methanol va 5 ml H 2SO4
96% cho vao bnh cau, un hoi lu cach thuy, khuay nhe
bang khuay t, dung dch c khuay trong 6 gi. Lay
dung dch nay em loc qua pheu loc hut chan khong, hon
hp phan ng hoa tan vao 400 ml Ethylacetat, sau o ra
vi 100 ml dung dch NaCl 15% (1 lan), 150 ml nc cat (3
lan), tiep tuc s dung co quay chan khong e loai bt
dung moi. San pham thu c em say chan khong
80oC en khoi lng khong oi, thu va can tho.

1 : Ong sinh han.


2 : Nhiet ke.
3 : Bnh cau.
4 : Hon hp phan
ng.
5 : May khuay t.

2
3
4
5

Hnh 4 : He thong un hoi lu.


Khao sat cac yeu to anh hng en qua trnh
tong hp :
- Thi gian phan ng : 4, 5, 6, 7 va 8 gi.
- Lng dung moi tham gia phan ng : 50, 75, 100,
125, 150 ml Methanol.
- Lng xuc tac tham gia phan ng : 3, 4, 5, 6, 7 ml
H2SO4 96%.

Hoc vien Nguyen Th Bch Tram


Trang 51

Luan van thac s


Sau khi khao sat tng yeu to, chon ra thong so
phu hp nhat. Thong so o c s dung e tiep tuc
khao sat yeu to tiep theo.

III.2. Tinh che Hesperetin.


Ket tinh lai trong ethanol :
Can 1 gam Hesperetin tho, hoa tan trong 50 ml Ethanol
800 them 0.2 gam than hoat tnh, un soi va khuay nhe
bang khuay t cho en khi tan hoan toan. Loc nong dung
dch bang giay loc. Dch qua loc c tiep tuc un soi
hoac s dung co quay chan khong e loai bt dung moi.
Sau o, e lanh dung dch, Heperetin se ket tinh lai. Loc
ap suat thap e thu tinh the Hesperetin va say chan
khong 800 C. em can Hesperetin thu c e tnh hieu
suat tinh che.

Hoc vien Nguyen Th Bch Tram


Trang 52

Luan van thac s


IV.

NGHIEN CU IEU
TRIACETYL HESPERETIN.

KIEN

TONG

HP

IV.1. Khao sat qua trnh tong hp Hesperetin


thanh Triacetyl Hesperetin.
Lay 0,2 gam Hesperetin tinh, 5 ml Anhydric acetic va 0.2
gam Natri acetat cho vao bnh cau sau o un hoi lu cach
cat, khuay nhe bang khuay t, dung dch c khuay soi
trong 1 phut. Lay dung dch nay em o vao hon hp
nc a, khuay eu roi e yen cho san pham ket tinh
xuong. Loc va ra lai that sach vi nc lanh tren pheu
loc Buchner. San pham thu c em say chan khong
80oC en khoi lng khong oi, thu va can tho.
Khao sat cac yeu to anh hng en qua trnh
tong hp :
- Thi gian phan ng : 1, 5, 10, 15 va 20 phut.
- Lng dung moi tham gia phan ng : 1, 2, 3, 4, 5
ml Anhydric acetic.
- Lng xuc tac tham gia phan ng : 0.025, 0.05,
0.075, 0.10, 0.125 gam Natri acetat.
Sau khi khao sat tng yeu to, chon ra thong so
phu hp nhat. Thong so o c s dung e tiep tuc
khao sat yeu to tiep theo.

IV.2. Tinh che Triacetyl Hesperetin.


Ket tinh lai trong Ethanol :
Can 1 gam Triacetyl Hesperetin tho, hoa tan trong 20 ml
Ethanol, un soi va khuay nhe bang khuay t cho en khi
tan hoan toan, loc nong dung dch bang giay loc, e yen
cho tinh the ket tinh, loc ap suat thap e thu tinh the
Triacetyl Hesperetin, sau o say chan khong 80 oC. em
can Triacetyl Hesperetin thu c e tnh hieu suat tinh
che.

Hoc vien Nguyen Th Bch Tram


Trang 53

Luan van thac s


V. NHAN DANH SAN PHAM.
V.1. Nhan danh Hesperidin.
Xac nh bang sac ky lp mong, nhiet o nong chay,
UV-Vis, IR, LC-MS :
V.1.1. Sac ky lp mong, o nhiet o nong
chay.
-

Sac ky lp mong :

Hoa tan mot t Hesperidin vao dung dch MeOH. Dch


MeOH va pha c cham len ban mong Silicagel chay vi
he dung moi n-butanol acid acetic - nc vi ty le 4 :1 : 5
(lay lp tren). Ban mong loai 25DC-Alufolien 20x20 cm,
Kiesegel 60 F254, Merk.
-

o nhiet o nong chay :

Chon ong mao quan thuy tinh co o day eu nhau,


ng knh 1-12 mm, dai 70 -100 mm, mot au han kn.
Ong phai that sach va kho, cho Hesperidin vao ong mao
quan, sau o xac nh iem chay bang may o iem
chay. iem chay c o 2 lan, ket qua lay trung bnh
cua 2 lan o iem chay. S dung may o iem chay
ELECTRONTHERMAL 9000 tai phong hp chat thien nhien
Vien Cong nghe Hoa hoc Viet Nam.
V.1.2. Xac nh bang pho UVVis.
Pha dung dch Hesperidin trong Methanol vi cac nong
o xac nh. Dung may UV 2450 Shimadzu tai Vien Cong
Nghe Hoa Hoc, o o hap thu dung dch trong vung bc
song t 200 - 800 nm. Quan sat pho, xac nh cc ai.
V.1.3. Xac nh bang pho IR .
Pho IR ghi tren may quang pho hong ngoai IR Vector
22 Bruker cua Vien Cong Nghe Hoa hoc.

Hoc vien Nguyen Th Bch Tram


Trang 54

Luan van thac s

V.1.4. Xac nh bang pho LC-MS.


Pho LC-MS c ghi tren he thong Sac ky long cao
ap LC 1090 Hewlett Parkard ghep khoi pho cua hang
MSD-Trap 1100 Aginent.

V.2. Nhan danh Hesperetin.


Xac nh bang sac ky lp mong, nhiet o nong chay,
UV-Vis, IR, HPLC :
V.2.1. Sac ky lp mong, o nhiet o nong chay.
Tng t phan V.1.1.
V.2.2. Xac nh bang pho UV-Vis.
Tng t phan V.1.2.
V.2.3. Xac nh bang pho IR.
Tng t phan V.1.3.
V.2.4. Xac nh bang pho HPLC.
Pho HPLC c ghi tren he thong Sac ky long cao
ap LC 1090 Hewlett Parkard.

V.3. Nhan danh Triacetyl Hesperetin.


Xac nh bang sac ky lp mong, nhiet o nong chay,
UV-Vis, IR, HPLC, NMR :
V.3.1. Sac ky lp mong, o nhiet o nong chay.
Tng t phan V.1.1.
V.3.2. Xac nh bang pho UVVis.
Tng t phan V.1.2.
V.3.3. Xac nh bang pho IR.
Tng t phan V.1.3.

Hoc vien Nguyen Th Bch Tram


Trang 55

Luan van thac s


V.3.4. Xac nh bang pho HPLC.
Tng t phan V.2.4.
V.3.5. Xac nh bang NMR.
Pho cong hng t hat nhan 1H-NMR, 13C-NMR ghi tren
may cong hng t hat nhan Bruker AC 200, tan so 500
MHz tai Vien Hoa hoc Ha Noi.

Hoc vien Nguyen Th Bch Tram


Trang 56

Luan van thac s


VI.

XAC NH HOAT
KHANG KHUAN.

TNH KHANG

NAM,

Cac mau c th hoat tnh khang khuan, khang


nam tai phong Sinh hoc thc nghiem Vien Hoa hoc cac
Hp chat thien nhien Vien KH & CN Viet Nam.

VI.1. Cac loai vi khuan, nam th nghiem.


Viec th nghiem c tien hanh tren cac chung
sau :
Vi khuan :
-

Vi khuan Gr(-) :
+ Escherichia coli,
+ Pseudomonas aeruginosa.

Vi khuan Gr(+) :
+ Bacillus subtillis ,
+ Staphylococcus aureus.

Nam :
-

Nam si :
+ Aspergillus niger,
+ Fusarium oxysporum.

Nam men :
+ Candida albicans,
+ Saccharomyces cerevisiae.

VI.2. Moi trng th nghiem.

Hoc vien Nguyen Th Bch Tram


Trang 57

Luan van thac s


Nam va vi khuan c duy tr trong moi trng dinh
dng : Saboraud dextrose broth va Trypease soya broth (TSB).

VI.3. Chat th nghiem.


-

Hesperidin,
Hesperetin,
Triacetyl Hesperetin.

VI.4. Tien hanh khao sat.


Chon phng phap tien hanh la phng phap hien
ai cua Vanden Bergher va Vlietlinck (nh phan 9 V.1)

Hoc vien Nguyen Th Bch Tram


Trang 58

Luan van thac s

Phan 3 :

KET QUA & BIEN


LUAN

Hoc vien Nguyen Th Bch Tram


Trang 59

Luan van thac s


I. TACH CHIET HESPERIDIN.
I.1. Tach chiet Hesperidin.

Hieu suat chiet Hesperidin trong Methanol:

(%) =

m'
* 100%
m

6.2170
* 100%
100

= 6.22%
m: khoi lng Hesperidin thu c (gam)
m : khoi lng vo qut em chiet
(gam)

I.2. Tinh che Hesperidin.


.

Hesperidin chiet xuat c em tinh che va ket tinh


nhieu lan trong Methanol. Kiem tra bang sac ky lp
mong cho thay co mot vet tron ep, vi Rf = 0.58.

Hieu suat tinh che Hesperidin :


Khoi lng Hesperidin tinh
H% = * 100 %
Khoi lng Hesperidin tho
= 86.40 %

II. NGHIEN CU IEU KIEN TONG HP


HESPERETIN
II.1. Khao sat qua trnh tong hp.

Hoc vien Nguyen Th Bch Tram


Trang 60

Luan van thac s


Tien hanh khao sat anh hng cua thi gian,
lng Methanol, acid H2SO4 96% len khoi lng cua san
pham phan ng :
II.1.1. Khao sat ve thi gian phan ng.
ieu kien th nghiem:
+ Khoi lng nguyen lieu
: 5,0000 gam
Hesperidin.
+ Lng dung moi tham gia phan ng : 100 ml MeOH , 5 ml
H2SO4 96%.
+ Nhiet o phan ng
: nhiet o soi cua
dung dch.
Bang 1 : Ket qua khao sat bien oi Hesperidin
thanh Hesperetin theo thi gian phan ng.
Thi gian (gi)
Khoi lng
Hesperetin thu
c (gam)

4
5
6
7
8
1.4190 1.8086 2.3616 2.3612 2.3614

o th 1 : Bieu dien s bien oi khoi lng san


pham Hesperetin theo thi gian.
Khoi lng (g)

2.5
2
1.5
1
0.5
0
0

10

Nhan xet :
Th
i gian (gi
)
- Thi gian phan
ng anh
hng kha ln en
phan ng.

Hoc vien Nguyen Th Bch Tram


Trang 61

Luan van thac s


-

T 3 gi en 6 gi lng san pham tang


tng oi ang ke, tuy nhien khi tiep tuc
tang them thi gian th khoi lng san pham
hau nh khong thay oi nen co the xem thi
gian phan ng trong 6 gi la toi u.
Sau khi lam tinh mot lan, qua ket qua sac ky
lp mong vi dung moi khai trien la n-BuOH :
AcOH : H2O = 4 :1 :5 (lay lp tren), tat ca cac
mau th nghiem eu cho mot vet, ieu nay
chng to phan ng xay ra thuan li, san
pham la Hesperetin. S dung ieu kien nay
e khao sat cac yeu to tiep theo.

II.1.2. Khao sat ve lng Methanol tham gia phan


ng.
ieu kien th nghiem:
-

Khoi lng nguyen lieu : 5,0000 gam


Hesperidin.
H2SO4 96%
: 5 ml.
Thi gian phan ng
: 6 gi.
Nhiet o phan ng
: nhiet o soi
cua dung dch .

Bang 2: Ket qua khao sat bien oi Hesperidin thanh


Hesperetin theo lng Methanol tham gia phan ng.
Lng dung moi
(ml)
Khoi lng
Hesperetin thu
c (gam)

50

75

100

125

150

1.7623

1.9680

2.361
6

2.361
5

2.360
5

Hoc vien Nguyen Th Bch Tram


Trang 62

Luan van thac s


o th 2 : Bieu dien khoi lng san pham Hesperetin theo
lng MeOH tham gia phan ng.
Khoi lng (g)

2.5
2
1.5
1
0.5
0
0

50

100

150

200

L ng dung mo
i (ml)

Nhan xet :
- Lng san pham thay oi theo lng Methanol.
Khi ta thay oi the tch cua Methanol t 50 -100
ml th khoi lng Hesperetin tang vot va at
gia tr cao nhat tai 100 ml. Sau o khoi lng
san pham hau nh khong thay oi khi ta tang
them lng MeOH.
- Khi tang them lng MeOH se giup qua trnh
hoa tan Hesperidin tot hn. Do vay, khi phan
ng het lng Hesperidin th khoi lng san
pham khong oi, khi ta tiep tuc tang lng
MeOH.
II.1.3.Khao sat ve lng H2SO4 96% tham gia phan
ng.
ieu kien th nghiem :
-

Khoi lng nguyen lieu : 5 gam Hesperidin.


Thi gian phan ng
: 6 gi.
Lng MeOH
:100 ml.

Hoc vien Nguyen Th Bch Tram


Trang 63

Luan van thac s


Bang 3 : Ket qua khao sat bien oi Hesperidin thanh
Hesperetin theo lng H2SO4 96%.
H2SO4 96%(ml)
Khoi lng
Hesperetin thu
c (gam)

3
1.7180

4
2.210
5

5
2.3616

6
2.361
1

7
2.361
6

o th 3 : Bieu dien khoi lng san pham Hesperetin theo


lng H2SO4 96% tham gia phan ng.

Khoi lng (g)

2.5
2
1.5
1
0.5
0
0

L ng acid (ml)

Nhan xet :
- Lng acid co anh hng rat ln en phan
ng, ong vai tro tac nhan cat mach.
- Lng san pham thay oi theo lng acid. Khi
tang lng acid 3 en 5 ml th khoi lng san
pham tang va at gia tr cao nhat tai 5 ml.
- Khi tang them lng acid (6 ml, 7 ml) th lng
san pham hau nh khong oi (v phan ng
a chuyen hoa het thanh Hesperitin) va vi
lng acid tren cung khong lam chay san
pham.

Ket luan :

Hoc vien Nguyen Th Bch Tram


Trang 64

Luan van thac s


Da tren cac ket qua phan ng tren, ta co
the chon ieu kien toi u cho phan ng tong hp
Hesperetin nh sau :
-

Khoi lng nguyen lieu : 5 gam Hesperidin.


Lng MeOH
: 100 ml.
Lng H2SO4 96%
: 5 ml.
Nhiet o phan ng
: tsoi cua dung

dch.

- Thi gian phan ng


- Lng san pham
Hesperetin.

: 6 gi.
2.3616

gam

Hieu suat phan ng tong hp Hesperetin :


Khoi lng Hesperetin thu c
610.55
H% = * * 100 %
Khoi lng Hesperidin em phan ng

302.27

= 95.40 %
610.55 = phan t lng Hesperidin
302.27 = phan t lng Hesperetin

II.2. Tinh che Hesperetin.


Hesperetin tong hp c em tinh che va ket tinh
nhieu lan trong Ethanol. Kiem tra bang sac ky lp mong
cho thay co mot vet tron ep, vi Rf = 0.93.

Hieu suat tinh che Hesperetin :


Khoi lng Hesperetin tinh
H% = * 100 %
Khoi lng Hesperetin tho
= 62.52 %

Hoc vien Nguyen Th Bch Tram


Trang 65

Luan van thac s


III.

NGHIEN CU IEU
TRIACETYL HESPERETIN.

KIEN

TONG

HP

III.1. Khao sat qua trnh tong hp Hesperetin


thanh Triacetyl Hesperetin.
Tien hanh khao sat anh hng cua thi gian,
lng (CH3CO)2O, lng CH3COONa len khoi lng cua san
pham phan ng :
III.1.1. Khao sat ve thi gian phan ng.
ieu kien th nghiem :
- Khoi lng nguyen lieu
Hesperetin.
- Lng dung moi (CH3CO)2O
- Lng CH3COONa
- Nhiet o phan ng
dung dch.

: 0.2000 gam
: 5 ml.
: 0.2000 gam.
: nhiet o soi cua

Bang 4 : Ket qua khao sat bien oi Hesperetin


thanh Triacetyl Hesperetin theo thi gian phan ng.
Thi gian (phut)
Khoi lng Triacetyl
Hesperetin thu c
(gam)

1
0.237
6

5
0.207
9

10
0.197
8

Hoc vien Nguyen Th Bch Tram


Trang 66

15
0.196
2

20
0.189
9

Luan van thac s


o th 4 : Bieu dien khoi lng san pham Triacetyl
Hesperetin theo thi gian.

Khoi lng (g)

0.25
0.2
0.15
0.1
0.05
0
0

10

15

20

25

Th
i gian (phu
t)

Nhan xet :
- Thi gian phan ng anh hng kha ln en
phan ng.
- Sau thi gian 1 phut th khoi lng san pham
at gia tr cao nhat.
- Tiep tuc tang thi gian (5, 10, 15, 20 phut) th
khoi lng san pham giam dan cho thay
cang keo dai thi gian phan ng th san
pham phan ng se giam do phan ng
chuyen dch theo chieu lam giam khoi lng
san pham.
- Sau khi lam tinh mot lan, qua ket qua sac ky
lp mong vi dung moi khai trien la n-BuOH :
AcOH : H2O = 4 :1:5 (lay lp tren), tat ca cac
mau th nghiem eu co mot vet, ieu nay
chng to phan ng xay ra thuan li, san
pham la Triacetyl Hesperetin. S dung ieu
kien nay e khao sat cac yeu to tiep theo.

Hoc vien Nguyen Th Bch Tram


Trang 67

Luan van thac s

III.1.2. Khao sat ve lng


phan ng.

(CH3CO)2O

tham gia

ieu kien th nghiem:


-

Khoi lng nguyen lieu : 0.2000 gam


Hesperetin.
Lng CH3COONa
: 5 ml.
Thi gian phan ng
: 1 phut.
Nhiet o phan ng
: nhiet o soi
cua dung dch .

Bang 5 : Ket qua khao sat bien oi Hesperetin thanh


Triacetyl Hesperetin theo lng (CH3CO)2O tham gia phan ng.
Lng (CH3CO)2O
(ml)
Khoi lng Triacetyl
Hesperetin thu c
(gam)

0.16
52

0.2124

0.237
6

0.237
3

0.236
9

Khoi lng (g)

o th 5 : Bieu dien khoi lng san pham Triacetyl


Hesperetin theo lng (CH3CO)2O tham gia phan ng.
0.25
0.2
0.15
0.1
0.05
0
0

L ng dung mo
i (ml)

Nhan xet :

Hoc vien Nguyen Th Bch Tram


Trang 68

Luan van thac s


-

Lng san pham thay oi theo lng (CH3CO)2O.


Khi ta thay oi the tch cua (CH3CO)2O t 1 - 3
ml th khoi lng Triacetyl Hesperetin tang vot
va at gia tr cao nhat tai 3 ml. Sau o khoi
lng san pham hau nh khong thay oi khi ta
tang them lng (CH3CO)2O.
Khi tang them lng (CH3CO)2O se giup qua
trnh phan ng xay ra theo chieu thuan. Tuy
nhien khi tang en mot nong o thch hp th
khoi lng san pham khong oi.

III.1.3. Khao sat ve lng CH3COONa


phan ng.

tham gia

ieu kien th nghiem:


-

Khoi lng nguyen lieu : 0.2000 gam


Hesperetin.
Lng (CH3CO)2O
: 3 ml.
Thi gian phan ng
: 1 phut.
Nhiet o phan ng
: nhiet o soi
cua dung dch .

Bang 6 : Ket qua khao sat bien oi Hesperetin thanh


Triacetyl Hesperetin theo lng CH3COONa tham gia phan
ng.
Lng CH3COONa
(gam)
Khoi lng Triacetyl
Hesperetin thu c
(gam)

0.02
5
0.15
27

0.05

0.075

0.10

0.125

0.175
9

0.237
6

0.237
0

0.237
2

Hoc vien Nguyen Th Bch Tram


Trang 69

Luan van thac s


o th 6 : Bieu dien khoi lng san pham Triacetyl
Hesperetin theo lng xuc tac CH3COONa tham gia phan
ng.

Khoi lng (g)

0.25
0.2
0.15
0.1
0.05
0
0

0.05

0.1

0.15

L ng xu
c ta
c (g)

Nhan xet :
- Lng san pham thay oi theo lng
CH3COONa. Khi ta thay oi lng CH3COONa t
0.025 - 0.075 gam th khoi lng Triacetyl
Hesperetin tang vot va at gia tr cao nhat tai
0.075 gam. Sau o khoi lng san pham hau
nh khong thay oi khi ta tang them lng
CH3COONa.
Ket luan :
Da tren cac ket qua khao sat tren, ta co
the chon ieu kien toi u cho phan ng tong hp
Triacetyl Hesperetin nh sau :
- Khoi lng nguyen lieu : 0.2000 gam Hesperetin.
- Lng CH3COONa
: 0.075 gam.
- Lng (CH3CO)2O
: 3 ml.
- Nhiet o phan ng
: tsoi cua dung dch.
- Thi gian phan ng
:1 phut.
- Lng san pham
:
0.2376
gam
Triacetyl
Hesperetin.

Hoc vien Nguyen Th Bch Tram


Trang 70

Luan van thac s

Hieu suat phan ng tong hp Triacetyl Hesperetin :

Khoi lng Triacetyl Hesperetin thu c


302.16
H% = * * 100 %
Khoi lng Hesperetin em phan ng

428

= 83.87 %
302.27 = phan t lng Hesperetin
428
= phan t lng Triacetyl Hesperetin

III.2. Tinh che Triacetyl Hesperetin.


Triacetyl Hesperetin sau khi c tao thanh em tinh che
va ket tinh nhieu lan trong Ethanol. Kiem tra bang sac ky
lp mong cho thay mot vet tron ep, vi R f = 0.93

Hieu suat tinh che Triacetyl Hesperetin :


Khoi lng Triacetyl Hesperetin tinh
H% = * 100 %
Khoi lng Triacetyl Hesperetin tho
= 93.81%

Hoc vien Nguyen Th Bch Tram


Trang 71

Luan van thac s


IV. NHAN DANH SAN PHAM.
IV.1 Nhan danh Hesperidin.
Hesperidin sau khi c tinh che nhieu lan bang
Methanol c s dung e nhan danh :
IV.1.1. Sac ky lp mong, T0nc.
-

Dang bot mn.


Mau sac : mau trang.
Sac ky lp mong vi dung moi khai trien : nbutanol : acid acetic : H2O = 4 :1 :5 (lay phan
tren) : cho mot vet tron mau vang nau.
Nhiet o nong chay (T0nc ) : 258 2600.

IV.1.2. Pho UV-Vis (Ethanol).


Ket qua pho UV-Vis cho thay co 2 mui cc ai tai :
(xem phu luc 1).
+ = 285 nm.
+ = 329 nm.
IV.1.3.Pho IR.
Ket qua pho IR cho thay : (xem phu luc 4).
-

Mui 3472 cm-1 : la dao ong cua lien ket OH


Mui 2917 cm-1 : la dao ong cua lien ket CH
Mui 1648 cm-1 : la dao ong cua lien ket
C=O
Mui 1606 cm-1 : la dao ong cua lien ket
C=C
Mui 1204 1277 cm-1 : la dao ong cua lien
ket OH (phenol)
Mui 1132 1183 cm-1 : la dao ong cua lien
ket C-O

Hoc vien Nguyen Th Bch Tram


Trang 72

Luan van thac s


IV.1.4. Pho LC MS : (xem phu luc 7,10,11,12)
Pho LC :
ieu kien phan tch :
-

: ZORBAX SB_C18, 4.6 x 150 mm, 5 m, (P/N


883975-902)
Pha ong
:
A : H2O 50%
B : MeOH 50%
Gradient : at 1 min
: 50% B
at 6 min
: 80% B
at 10 min
: 100%
Toc o
: 1 ml/min.
Nhiet o : 400C.
au do : DAD 254 nm, bandwith : 4nm, Ref : off.
Cot

Ket qua cho thay o sach cua mau : 94.48% (xem


phu luc 7)
Pho MS :
ieu kien phan tch :
+ Dung moi
: Acetonitril.
+ Nhiet o cua nguon tao ion : 3500C.
+ Che o phan tch negative : [M - H+ ].
Ket qua (xem phu luc 11) cho : [M - H+ ] = 609.
Bien luan :
-

Mau Hesperidin sau khi lam tinh co nhiet o


nong chay 2580C. Ket qua sac ky lp mong
vi dung moi khai trien : n-butanol : acid acetic :
H2O ( 4 :1 :5 lay phan tren) cho mot vet tron
mau vang nau.
Pho UV-Vis co mui hap thu cc ai = 285
nm va = 329 nm, co bang II trong vung (275
295 nm), bang I trong vung (300 330 nm) ac
trng cho 2 he thong benzoyl va cinamoyl cua
vong A va B cua flavanon.

Hoc vien Nguyen Th Bch Tram


Trang 73

Luan van thac s


Ket qua so sanh iem chay, pho UV-Vis cua
Hesperidin dung nhan danh va Hesperidin trong tai
lieu :
Chat khao sat
Hesperidin / nhan
danh
Hesperidin / tai
lieu
-

iem chay
(0C)
258 260

285

329

258 262

283

326

max (nm)

Pho IR cho cac mui hap thu ac trng cua cac


nhom OH, C=O, C=C va C-H cua hp chat
hydrocacbon.
Pho LC MS :
+
Ket qua pho HPLC cho thay o sach cua
mau : 94.48%.
+ Khoi Pho MS cho ket qua [M H +] = 609, tng
ng vi m/z = 610 la phan t lng cua
Hesperidin.

Cac ket qua nhan danh tren so vi cac so lieu ve


Hesperidin cho phep nhan nh san pham chiet tach co
c chnh la Hesperidin.

IV.2. Nhan danh Hesperetin.


Hesperetin sau khi c tinh che nhieu lan bang
Ethanol c s dung e nhan danh :
IV.2.1. Sac ky lp mong, T0nc.
-

Dang tinh the hnh luc giac.


Mau sac : mau trang.
Sac ky lp mong vi dung moi khai trien : nbutanol : acid acetic : H2O = 4 :1 :5 (lay phan
tren) : cho mot vet tron mau vang nau.
Nhiet o nong chay (T0nc ) : 227 2300C.

IV.2.2. Pho UV-Vis (Methanol).

Hoc vien Nguyen Th Bch Tram


Trang 74

Luan van thac s


Ket qua pho UV-Vis cho thay co 1 mui cc ai tai :
(xem phu luc 2)
+ = 288 nm.
IV.2.3.Pho IR
Ket qua pho IR cho thay : (xem phu luc 5).
-

Mui 3498 cm-1 : la dao ong cua lien ket


OH.
Mui 3116 cm-1 : la dao ong cua lien ket
CH.
Mui 1641 cm-1 : la dao ong cua lien ket
C=O.
Mui 1508 cm-1 : la dao ong cua lien ket
C=C vong thm.
Mui 1176 cm-1 : la dao ong cua lien ket
C-O.

IV.2.4. Pho HPLC.(phu luc 8).


ieu kien phan tch :
-

: ZORBAX SB_C18, 4.6 x 150 mm, 5 m, (P/N


883975-902)
Pha ong
:
A : H2O 50%
B : MeOH 50%
Gradient : at 1 min
: 50% B
at 6 min
: 80% B
at 10 min
: 100%
Cot

Toc o
: 1 ml/min.
Nhiet o : 400C.
au do : DAD 254 nm, bandwith : 4nm, Ref : off.

Ket qua cho thay o sach cua mau : 98.14% (xem


phu luc 8)
Bien luan :

Hoc vien Nguyen Th Bch Tram


Trang 75

Luan van thac s


-

Mau Hesperetin sau khi lam tinh co nhiet o


nong chay 2270C. Ket qua sac ky lp mong
vi dung moi khai trien : n-butanol : acid acetic :
H2O ( 4 :1 :5 lay phan tren) cho mot vet tron
mau vang nau.
Pho UV-Vis co mui hap thu cc ai = 288
nm, co bang II trong vung (275 295 nm), bang I
trong vung (300 330 nm) ac trng cho 2 he
thong benzoyl, cinamoyl cua vong A va B cua
flavanon.
Ket qua so sanh iem chay, pho UV-Vis cua
Hesperetin dung nhan danh va Hesperetin trong
tai lieu :
Chat khao sat
Hesperetin / nhan
danh
Hesperetin / tai
lieu

iem chay
(0C)
227 230

max (nm)

226 228

288
288

Pho IR cho cac mui hap thu ac trng cua cac


nhom OH, C=O, C=C va C-H cua hp chat
hydrocacbon.
Ket qua pho HPLC cho thay o sach cua mau :
98.14%.

Cac ket qua nhan danh tren so vi cac so lieu ve


Hesperetin cho phep nhan nh san pham tong hp chnh
la Hesperetin.

IV.3. Nhan danh Triacetyl Hesperetin.


Triacetyl Hesperetin sau khi c tinh che nhieu lan
bang Ethanol c s dung e nhan danh :
IV.3.1. Sac ky lp mong, T0nc.
-

Dang bot vo nh hnh.

Hoc vien Nguyen Th Bch Tram


Trang 76

Luan van thac s


-

Mau sac : mau trang .


Sac ky lp mong vi dung moi khai trien : nbutanol : acid acetic : H2O = 4 :1 :5 (lay phan
tren) : cho mot vet tron mau vang nau.
Nhiet o nong chay (T0nc ) : 135 1390C.

IV.3.2. Pho UV-Vis (Methanol).


Ket qua pho UV-Vis cho thay co 2 mui cc ai tai : (xem
phu luc 3).
+ = 315 nm.
+ = 261 nm.
IV.3.3.Pho IR.
Ket qua pho IR cho thay : (xem phu luc 6).
-

Mui 1771 cm-1 : la dao ong cua lien ket


C=O ester.
Mui 1692 cm-1 : la dao ong cua lien ket
C=C vong thm.
Mui 1436 cm-1 : la dao ong bien dang bat
oi xng cua lien ket CH3.
Mui 1373 cm-1 : la dao ong bien dang oi
xng cua lien ket CH3.
Mui 1192 cm-1 : la dao ong cua lien ket
C-O.

IV.3.4.Pho HPLC. (Xem phu luc 9).


Ket qua cho thay o sach cua mau : 84.34%
IV.3.5. Pho NMR.
Ket qua 1H-NMR (xem phu luc 13, 14, 15).
Bang 7: 1H-NMR cua Triacetyl Hesperetin.
3

Hoc vien Nguyen Th Bch Tram


Trang 77

Luan van thac s


V tr proton
- CH3 (3 x
OAc)

o dch chuyen hoa hoc (,ppm)


2.264 ( x 3H, s)
2.273 ( x 3H, s)
2.295 ( x 3H, s)
3.794 ( x 3H, s)
3.300 3.240 (1H, dd, J = 13Hz va J =
16.5Hz)
2.736 2.697 (1H, dd, J = 3Hz va J =
16.5Hz)
5.630 5.604 (1H, dd, J = 13Hz va J =
3Hz)
6.691 6.687 (1H, d, J = 2Hz)
6.875 6.871 (1H, d, J = 2Hz)
7.186 7.169 (1H, d, J = 8.5Hz)
7.304 7.300 (1H, d, J = 2Hz)
7.422 7.401 (1H, dd, J = 2Hz va J =
8.5Hz)

- OCH3
H3a
H3b
H2
H8
H6
H5
H2
H6

Ket qua

13

Bang 8:

C-NMR cua Triacetyl Hesperetin.

13

C-NMR (xem phu luc16, 17, 18)

V tr C

Loai C

- OCOCH3
- OCOCH3

- CH3

3
- OCH3
2
8
6
10
5
2
6
1
3
5
4

C t cap C
=O
- CH2
- CH3
- CH
- CH
- CH
- CH
- CH
- CH
C t cap
C t cap

o chuyen dch hoa hoc


( , ppm )
20.445
20.875
20.938
168.484
168.690
168.828
43.795
56.066
78.362
109.353
110.905
111.623
112.869
121.641
125.789
130.722
139.338

Hoc vien Nguyen Th Bch Tram


Trang 78

Luan van thac s


7
9
4

C
C
C
C
C

t cap
t cap
t cap
t cap
t cap
C=O

150.821
151.345
155.814
162.819
189.350

Bien luan :
-

Mau Triacetyl Hesperetin sau khi lam tinh co nhiet


o nong chay 1350C. Ket qua sac ky lp
mong vi dung moi khai trien : n-butanol : acid
acetic : H2O ( 4 :1 :5 lay phan tren) cho mot vet
tron mau vang nau.
Pho UV-Vis co mui hap thu cc ai = 261
nm va = 315 nm, co bang II trong vung (275
295 nm), bang I trong vung (300 330 nm) ac
trng cho 2 he thong benzoyl va cinamoyl cua
vong A va B.
Ket qua so sanh iem chay cua Triacetyl
Hesperetin dung nhan danh va Triacetyl Hesperetin
trong tai lieu :
Chat khao sat
Triacetyl Hesperetin / nhan
danh
Triacetyl Hesperetin / tai
lieu

Hoc vien Nguyen Th Bch Tram


Trang 79

iem chay
(0C)
135 139
139 141

Luan van thac s


-

Pho IR cho cac mui hap thu ac trng cua cac


nhom C=O, C=C va C-H cua hp chat
hydrocacbon.
Pho 13C-NMR cho thay phan t co 22 nguyen
t carbon, gom :
+ 11 carbon t cap (1 nhom C=O cua vong
va 3 nhom C=O cua ester)
+ 6 nhom CH
+ 1 nhom CH2
+ 4 nhom CH3
Ket qua pho 1H-NMR cho thay phan t co 20
nguyen t Hidro, gom :
+ 5 nguyen t H cac v tr H 2, H6, H5, H8,
H6 at trng cho C-H cua vong thm A, B.
+ 9 nguyen t H cua 3 nhom - CH 3 trong goc
ester
+ 3 nguyen t H cua nhom CH 3 trong goc
ether
+ 2 nguyen t H cua nhom CH2
+ 1 nguyen t H cua nhom CH
Ket qua pho HPLC cho thay o sach cua mau :
84.34%

Ket hp cac ket qua T0nc, UV-Vis, IR, HPLC, 1H-NMR, 13CNMR cho phep ket luan san pham tong hp c phu
hp cong thc phan t C22H20O9 cua Triacetyl Hesperetin.
Cong thc cau tao cua Triacetyl Hesperetin tong hp c
nh sau :

O
O C CH3
H 2' 3'
O CH3
H
4'
1
1'
H3C C O 7
9 O
H 6' 5' H
8
2
O
6 5
3
Hb H
10 4
H
H
H3 C C O
O a
O

Hoc vien Nguyen Th Bch Tram


Trang 80

Luan van thac s


V. XAC NH HOAT TNH KHANG NAM, KHANG
KHUAN. (xem phu luc 19).
Chung toi tien hanh th nghiem tnh khang nam
khang khuan cua Hesperidin, Hesperetin va Triacetyl
Hesperetin nham xac nh va so sanh hoat tnh sinh hoc
cua cac san pham nay. Viec th nghiem c tien
hanh theo phng phap hien ai cua Vanden Bergher va
Vlietlinck.
Bang 9 : Ket qua xac nh tnh khang nam khang
khuan.
Vi khuan

E.coli
P. aeruginosa
B.subtilis
S. aureus
Nam

Na
Asp.niger
m
F.oxysporu
mo
m
c
Na C.albicans
m S.cerevisiae
me
n

Nong o c che toi thieu (MIC


: g/ml)
Hesperid Hesperet
Triacetyl
in
in
Hesperetin
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
50
(-)
25
50
Nong o c che toi thieu (MIC :
g/ml)
Hesperid Hesperet
Triacetyl
in
in
Hesperetin
(-)
(-)
(-)
50
50
(-)
(-)
(-)
(-)
(-)
(-)
(-)

Nhan xet :

Hoc vien Nguyen Th Bch Tram


Trang 81

Luan van thac s


-

Hesperidin khang c nam F.oxysporum.


Hesperetin khang c vi khuan S.aureus va nam
F.oxysporum.
Triacetyl Hesperetin khang c 2 vi khuan
B.subtillis va S.aureus.

Hoc vien Nguyen Th Bch Tram


Trang 82

Luan van thac s

Phan 4:

KET LUAN

Hoc vien Nguyen Th Bch Tram


Trang 83

Luan van thac s


Trong nhng nam gan ay, cac nha khoa hoc tren
the gii cung nh Viet Nam ang tap trung ngien cu
tm kiem cac loai thuoc cha benh, cac phu gia dung
trong thc pham, my pham co nguon goc t thien
nhien e ap ng nhu cau cham soc sc khoe cho con
ngi ngay mot nang cao. Tren c s o, chung toi
thc hien e tai nay xuat phat t vo cua cay qut,
mot loai cay co san va trong nhieu Viet Nam, tong
hp nen mot so hp chat e gop phan vao lnh vc
nghien cu cha benh Viet Nam.
Qua qua trnh nghien cu, chung toi a co c nhng
ket qua nh sau :
1. Tach chiet Hesperidin, tien hanh thuy phan ra
Hesperetin. Qua trnh nay c thc hien vi hieu
suat cao, san pham tng oi tinh khiet (Hesperidin :
94.48%, Hesperetin : 98.14%).
2. Xac nh c ieu kien toi u e tong hp
Hesperetin.
3. Xac nh c ieu kien toi u e tong hp Triacetyl
Hesperetin.
4. Bang cac phng phap phan tch hoa ly hien ai
nh o nhiet o nong chay, UV, IR, HPLC, LC-MS, 1HNMR, 13C-NMR chung toi a xac nh c cau truc
cua san pham tong hp.
5. Ket qua xac nh hoat tnh khang khuan, khang
nam cho thay : Hesperetin va Triacetyl Hesperetin co
hoat tnh manh hn so vi Hesperidin.
ong thi vi ket qua nay, chung ta co the tiep
tuc
phat trien nghien cu tong hp, th
nghiem
chuyen sau cac hp chat chiet xuat va dan xuat cua
chung, khai tac toi u cac hoat tnh sinh hoc nham gop
phan vao viec tong hp cac loai thuoc phong chong,
cha tr cac benh nan y Viet Nam cung nh tren the
gii.
Cac kien ngh cho cac nghien cu tiep theo :

Hoc vien Nguyen Th Bch Tram


Trang 84

Luan van thac s


1. Tiep tuc nghien cu tong hp them mot so
dan xuat khac t Hesperetin cung nh t mot
vai flavonoid khac.
2. Th nghiem them tren cac chung vi khuan, vi
nam khac e co c mot cach nhn tong
quat hn ve hoat tnh cua cac san pham.
3. Th tac ong tren cac loai ong vat nh
chuot, tho
4. ac biet, tien hanh nghien cu tac dung cua
cac chat nay tren cac dong te bao ung th.
Vi viec kien ngh mot so nghien cu tiep theo
e tai nay, chung toi hy vong co the gop phan tao ra
cac san pham co the ng dung phuc vu trong cac lnh
vc y te, thc pham, my pham nc ta.

Hoc vien Nguyen Th Bch Tram


Trang 85

Luan van thac s


TAI LIEU THAM KHAO
[1].

Nguyen Van an, Nguyen Viet Tu, Phng phap


nghien cu hoa hoc cay thuoc, Nha xuat ban Y
hoc, 1985.

[2].

Nguyen Kim Phi Phung, Cac phng phap nhan danh,


trch ly, co lap cac hp chat hu c, Hoc phan cao
hoc trng ai hoc Khoa hoc T nhien, 2000 - 2001.

[3].

Nguyen Ngoc Hanh, Tach chiet va co lap hp chat


t nhien, Giao trnh cao hoc, 2002.

[4].

Dc ien Viet Nam III, NXB Y hoc, 2002.

[5].

o Tat Li, Nhng cay thuoc va v thuoc Viet Nam,


Nha xuat ban Y hoc, 2001.

[6].

Nguyen Hng Thao, Vo Van Leo, Ngo Thu Van,


Tran Hung, Khao sat Flavonoid t vo qut, Tap ch
Dc hoc - so 5/2001.

[7].

ai Duy Ban, L th Cam Van, ai Th Ngan Ha,


ai Th Hang Nga, Phong chong ung th, NXB Y hoc,
1998.

[8].

Nguyen nh Chc; Ngo Tuan Ky, Sach tra cu hoa


sinh, NXB Khoa hoc Ky Thuat, 1984.

[9].

Nguyen c Minh, Tnh khang khuan cua Cay Thuoc


Viet Nam, Nha xuat ban Y hoc, 1975.

[10]. Nguyen c Lng, Cong nghe Vi sinh vat, Trng


ai hoc Bach Khoa Tp.HCM,1996.
[11]. Nguyen c Lng, Pham Minh Tam, Ve sinh an toan
thc pham, ai hoc Ky Thuat Tp.HCM.
[12]. Thuyet minh th hoat tnh sinh hoc, phong sinh hoc
thc nghiem-Vien Hoa hoc cac hp chat thien
nhien-Vien KH & CN Viet Nam.

Hoc vien Nguyen Th Bch Tram


Trang 86

Luan van thac s


[13]. Davicco Marie-Jeanne (FR); Horcajada Marie Noelle (FR);
Morad Christine (FR); Coxam Veronique (FR), Use of
Hesperidin or one of its derivatives for Making a madicine for
bone formation stimulation, Agronomique Inst Nat Rech (FR),
2004-01-08.
[14]. Bok Song Hae (KR); Son Kwang Hee (KR); Jeong Tae Sook
(KR); Kwon Byoung Mog (KR); Kim Young Kook (KR); Choi Doil
(KR); Kim Sung Uk; Bae Ki Hwang Ingyu (KR); Moon Surk Sik
(KR); Kwon Young Kook (KR); Ahn Jung Ah (KR); Lee Eun Sook
(KR), Hesperidin and Hesperetin as 3- Hydroxy- 3Methylglutaryl CoA (HMG-CoA) reductase inhibitor, Korea inst
science technology (KR), 2004-03-25.
[15]. Hesperidin,
www.phytochemicals.info/phytochemicals
/hesperidin.php-8k-.
[16]. Hesperidin,
info.com/hesperidin.html-8k.
[17]. Hesperidin,
txtingred.

www.nutritional-supplement-

www.enzy.com/products/searchresults.asp?

[18]. Hesperetin,
www.pdrhealth.com/drug_info/nmdrugprofiles/nutsupdrugs/h
es_0318.shtml-11k- .
[19]. Robert E.Wingard, Palo, Calif, Conversion of Hesperidin into
Hesperetin, Pritchett et al., J. Amer. Chem. Soc., 68, 2018
(1946). Looker et al., J. Org. Chem., 25, 1829 (1960).
[20]. Song Hae Bok (KR); Tae Sook Jeong (KR); Myung Choi (KR),
Hesperidin and Hesperetin as inhibitor of Acyl CoA
cholesterolo- acyltransferase, inhibitting the accumlation of
macrophage-lipid complex on the arterial endothelium, and
preventing or treating hepatic diseases in a mammal, Korea
Inst Science Technology (KR), 200012-27.

Hoc vien Nguyen Th Bch Tram


Trang 87

Luan van thac s


[21]. Richard Mayer, Possible Cancer-fighting Properties Found in
Certain Bioflavonoids, U.S. Horticultural Research Laboratory,
August 9, 2000.
[22]. Miyake Toshio (JP); Yumoto Takashi(JP), Enzyme-reated
Hesperidin, Producing the same and Methol of Using
Enzyme-treated Hesperidin, Hayashibara Biochem Lab (JP),
1998-02-25.
[23]. Ji Young Kim, Kyung Jin Jung, Jae Sue Choi, Hae Young Chung,
Hesperetin : a Potent Antioxidant Againt Peroxynitrite, Taylor
& Francis, Volume 38, Number 7 July, 2004.
[24]. Hiroshge Chiba, Mariko Uehara, Xinxiang Wang, Ritsuko
Masuyama, Kazuharu suzuki, Kazuki Kanazawa, Yoshiko
Ishimi, Hesperidin, a Citrus flavonoid, Inhibits Bone Loss and
Decreases Serum and Hepatic Lipids in Ovariectomized Mice,
The
American
Society
for
Nutritional
Sciences
J.
Nutr.133:1892-1897, June 2003.
[25]. Warren Raphael (US); Akadiri Adebolat (US), Methods of
Using Hesperetin for Sebum Control and Treatment of Acne,
Procter and Gamble (US), 19961224.
[26]. T.A. Geissman, The Chemistry of Flavonoid Compounds,
Pergamon Press Oxford-London-New York-Paris, 1962.

Hoc vien Nguyen Th Bch Tram


Trang 88

Luan van thac s

Phan 5 :

PHU LUC

Hoc vien Nguyen Th Bch Tram


Trang 89

Luan van thac s

Phu luc 1: Pho UV-Vis cua Hesperidin.

Hoc vien Nguyen Th Bch Tram


Trang 90

Luan van thac s

Phu luc 2 : Pho UV-Vis cua Hesperetin.

Hoc vien Nguyen Th Bch Tram


Trang 91

Luan van thac s

Phu luc 3 : Pho UV-Vis cua Triacetyl Hesperetin.

Hoc vien Nguyen Th Bch Tram


Trang 92

Luan van thac s

Phu luc 4 : Pho IR cua Hesperidin.

Hoc vien Nguyen Th Bch Tram


Trang 93

Luan van thac s

Phu luc 5 : Pho IR cua Hesperetin.

Hoc vien Nguyen Th Bch Tram


Trang 94

Luan van thac s

Phu luc 6 : Pho IR cua Triacetyl Hesperetin.

Hoc vien Nguyen Th Bch Tram


Trang 95

Luan van thac s

Phu luc 7 : Pho HPLC cua Hesperidin.

Hoc vien Nguyen Th Bch Tram


Trang 96

Luan van thac s

Phu luc 8 : Pho HPLC cua Hesperetin.

Hoc vien Nguyen Th Bch Tram


Trang 97

Luan van thac s

Phu luc 9 : Pho HPLC cua Triacetyl Hesperetin.

Hoc vien Nguyen Th Bch Tram


Trang 98

Luan van thac s

Phu luc 10 : Khoi pho MS cua Hesperidin.

Hoc vien Nguyen Th Bch Tram


Trang 99

Luan van thac s

Phu luc 11 : Khoi pho MS cua Hesperidin.

Hoc vien Nguyen Th Bch Tram


Trang 100

Luan van thac s

Phu luc 12 : Khoi pho MS cua Hesperidin.

Hoc vien Nguyen Th Bch Tram


Trang 101

Luan van thac s

Phu luc 13 : Pho 1H-NMR cua Triacetyl Hesperetin.

Hoc vien Nguyen Th Bch Tram


Trang 102

Luan van thac s

Phu luc 14 : Pho cua 1H-NMR Triacetyl Hesperetin.

Hoc vien Nguyen Th Bch Tram


Trang 103

Luan van thac s


Phu luc 15 : D lieu pho
Hesperetin.

H-NMR cua Triacetyl

Hoc vien Nguyen Th Bch Tram


Trang 104

Luan van thac s

Phu luc 16 : Pho

13

C-NMR cua Triacetyl Hesperetin.

Hoc vien Nguyen Th Bch Tram


Trang 105

Luan van thac s

Phu luc 17 : Pho

13

C-NMR cua Triacetyl Hesperetin.

Hoc vien Nguyen Th Bch Tram


Trang 106

Luan van thac s


Phu luc 18 : D lieu pho
Hesperetin.

13

C-NMR cua Triacetyl

Hoc vien Nguyen Th Bch Tram


Trang 107

Luan van thac s

Phu luc 19 : Phieu tra li ket qua hoat tnh khang vi sinh
vat kiem nh.

Hoc vien Nguyen Th Bch Tram


Trang 108

Luan van thac s

TOM TAT
Tren c s Hesperidin, mot loai flavonoid co trong
nhieu loai cay, ac biet vi ham lng rat ln trong vo
qut, mot loai cay co san va trong nhieu Viet Nam,
chung toi thuy phan ra Hesperetin. Hesperetin cung la
mot loai flavonoid ang c nghien cu kha nhieu tren
the gii v tac dung cua no len nhieu loai benh khac
nhau. e nang cao hieu qua s dung cua cac hp chat
thien nhien cua Viet Nam trong nganh y te, chung toi
tien hanh thc hien e tai nay e tong hp mot vai
dan xuat cua Hesperetin, xet nghiem tnh khang nam,
khang khuan cua chung.
Ket qua thc hien e tai:
1. Tach chiet Hesperidin va tong hp c Hesperetin
vi hieu suat cao, tng oi tinh khiet.
2. Xac nh ieu kien toi u e tong hp Hesperetin.
3. Xac nh ieu kien toi u e tong hp Triacetyl
Hesperetin.
4. Bang cac phng phap phan tch hoa ly hien ai
nh o nhiet o nong chay, UV, IR, HPLC, LC-MS, 1HNMR, 13C-NMR chung toi a xac nh c cau truc
cua san pham tong hp.
5. Ket qua xac nh hoat tnh khang nam, khang
khuan cho thay: Hesperetin va Triacetyl Hesperetin co
hoat tnh manh hn so vi Hesperidin.
Ket qua nghien cu cho thay ta thu c san pham
co hieu suat ln, o tinh khiet kha cao, co the s dung
cac ket qua nay trong cac nghien cu tiep theo.

Hoc vien Nguyen Th Bch Tram


Trang 109

Luan van thac s

ABSTRACT
On the basis of Hesperidin, a type of flavonoid existing in a
majority of plants, particularly in citrus skin, with high content
which is present and grows in many locations in Vietnam,
hydrolysis was realised to produce Hesperetin. Hesperetin is also
a type of flavonoid and is a substance on which many studies
have been conducted worldwide because of its effect on many
different diseases. In order to improve the effectiveness of
Vietnam natural compounds in the health sector, this research has
been conducted to synthesize Hesperetin derivatives, to test their
antibacterial and antifungal characteristics.
Research results:
1. Hesperidin and Hesperetin were extracted and
synthesized with high yield and in relatively high purity.
2. Optimized conditions for the synthesis of Hesperetin
were found.
3. Optimized conditions for the synthesis of Triacetyl
Hesperetin were found.
4. Using physical chemistry analyses such as melting
point, UV, IR, HPLC, LC-MS, 1H-NMR, 13C-NMR the
synthesized products structure was determined.
5. The
results
of
antibacterial
and
antifungal
characteristics testing demonstrated that Hesperetin,
Triacetyl Hesperetin has stronger antibacterial and
antifungal activities than Hesperidin.
Research results indicate that products were obtained with
high yield and in very high purity and the results should be used
for the following research steps.

Hoc vien Nguyen Th Bch Tram


Trang 110

Luan van thac s

LI CAM N
Con xin cam n ba ma, anh ch, nhng ngi than
yeu nhat.
Em xin chan thanh cam n :
-

Thay Nguyen Cu Khoa a tan tnh hng dan, tao


moi ieu kien thuan li cho em trong thi gian thc
hien luan an,
Thay Chu Pham Ngoc Sn, thay Bui Tho Thanh, thay
Nguyen Cong Hao, co Nguyen Ngoc Sng va cac
thay co thuoc Vien Cong nghe Hoa hoc a tan
tnh day do, giup em trong qua trnh hoc tap.
Phong Quan Ly Khoa Hoc- Sau ai Hoc ai Hoc Can
Th,
Cac thay co trong Hoi ong Bao ve Luan An,
Ch Hoang Th Kim Dung cung cac anh ch phong
Cong nghe Hu c cao phan t Vien Cong nghe
Hoa hoc a nhiet tnh ch bao, giup trong suot
thi gian lam luan an.

Va tat ca cac ban a giup , ong vien toi trong


suot qua trnh hoc tap va thc hien luan an.
Xin chan thanh cam n.

Hoc vien Nguyen Th Bch Tram


Trang 111

Luan van thac s


BANG TOM TAT QUA TRNH HOC TAP, CONG TAC
VA
HOAT ONG KHOA HOC KY THUAT
Ho Ten
: NGUYEN TH BCH TRAM
Lp
: Cao hoc Hoa hoc
Khoa
: 2003
Ngay sinh : 07 / 08 / 1979
Ni sinh
: Quang Nam
C quan cong tac
:
Tot nghiep ai hoc : C nhan S pham nganh Hoa
hoc nam 2001
Trng ai hoc S pham Hue.
Thi gian va ni cong tac t ngay ra trng en nay :
2001-2002
: Giao vien Trng PTTH Chu Van An, ai
ong, ai Loc, Quang Nam.
TOM TAT QUA TRNH HOC TAP VA NGHIEN CU KHOA
HOC
1. Boi dng kien thc trong va ngoai nc (t sau
tot nghiep ai hoc) :
2. Cac cong trnh nghien cu co lien quan en e
tai :
3. Luan van cao hoc :
Ten e tai : Tach chiet Hesperidin, tong hp
Hesperetin, Triacetyl Hesperetin va khao sat hoat tnh
khang nam, khang khuan cua chung
Can bo hng dan chnh : Ts NGUYEN CU KHOA Vien
Cong nghe Hoa hoc.
Can Th, ngay 18 thang
10 nam 2005.

Ngi khai

NGUYEN
TRAM
Y kien nhan xet cua n v quan ly
Tong so mon a hoc

Hoc vien Nguyen Th Bch Tram


Trang 112

TH

mon

BCH

Luan van thac s


trnh

Tong so n v hoc trnh a hoc

n v hoc

iem trung bnh chung hoc tap :


/ 10
a hoan thanh chng trnh hoc tap
QLKH TSH

Hoc vien Nguyen Th Bch Tram


Trang 113

Phong

Luan van thac s

MUC LUC
Trang
LI CAM N
NHIEM VU LUAN AN
TOM TAT
MUC LUC
DANH MUC CAC S O, BANG
DANH MUC CAC HNH, O TH
DANH MUC PHU LUC
M AU..........................................................................................................................1
I. FLAVONOID.....................................................................................................................3
I.1. Phan bo. [1],[3].........................................................................................................3
I.2. Cau truc hoa hoc. [1],[2],[3].................................................................................3
I.3. Phan loai flavonoid.[1],[3]........................................................................................4
I.3.1. Flavon.................................................................................................................4
I.3.2. Flavanon................................................................................................................5
I.3.4. Dihydroflavonon...................................................................................................6
I.3.5. Chalcon.................................................................................................................7
I.3.6. Dihydrochalcon...................................................................................................7
I.3.7. Auron..................................................................................................................8
I.3.8. Anthoxyanidin (2-phenylbenzo pyrilium)............................................................9
I.3.9. Leucoanthoxyanidin..............................................................................................9
I.3.10. Isoflavon...........................................................................................................10
I.3.11. Rotenoid............................................................................................................10
I.3.12. Neoflavonoid...................................................................................................11
I.3.13. Biflavonoid.....................................................................................................11
I.4. Vai tro cua Flavonoid.[1],[3]................................................................................12
I.4.1. Vai tro cua Flavonoid trong cay. ................................................................12
I.4.2. Vai tro cua flavonoid trong y hoc................................................................13
II. HESPERIDIN. ................................................................................................................17
II.1. Cac loai cay co cha Hesperidin......................................................................17
II.2. Cau tao cua Hesperidin.[8],[13],[15],[16]...........................................................18
II.3. Tnh chat vat ly.[6],[8].......................................................................................18
II.4. ng dung cua Hesperidin...................................................................................19
III. HESPERETIN................................................................................................................23
III.1. Cau tao.[8],[18]...................................................................................................23
III.2. Tnh chat vat ly.[6],[8].......................................................................................23
.........................................................................................................................................23
III.3. Phan ng ieu che.[19]....................................................................................24
III.4. ng dung cua Hesperetin..................................................................................24
IV. TRIACETYL HESPERETIN. [8],[26]..........................................................................26
IV.1. Cau tao................................................................................................................26

Hoc vien Nguyen Th Bch Tram


Trang 114

Luan van thac s


IV.2 . Tnh chat vat ly.................................................................................................26
IV.3. ieu che...............................................................................................................27
IV.4. ng dung.............................................................................................................27
V. XAC NH HOAT TNH KHANG NAM, KHANG KHUAN....................28
V.1. Cac phng phap nh tnh phat hien tnh khang khuan..............................28
V.1.1. Phng phap tron thuoc vao moi trng thach.[9]. .............................28
V.1.2. Phng phap uc lo trong moi trng thach. [9]...................................28
V.1.3. Phng phap tham giay. [9].......................................................................28
V.1.4. Phng phap ong tru cua Heatley. [9]..................................................29
V.1.5. Phng phap ao ranh cua Fleming (1932). [9]......................................29
V.1.6. Phng phap vien nen ang Van Ng (1956). [9]..............................30
V.1.7. Phng phap th nhng chat khang khuan bay hi. [9].......................30
V.1.8. Phng phap sac ky khang khuan. [9]...................................................31
V.1.9. Phng phap hien ai cua Vanden Benergher va Vlietlinck (1994).[12 ].
......................................................................................................................................31
V.2. Cac loai vi khuan, vi nam s dung e xac nh hoat tnh.[10],[11]..........32
V.2.1. Vi khuan........................................................................................................32
V.2.2. Nam.................................................................................................................33
NOI DUNG NGHIEN CU :......................................................................................36
A. PHNG PHAP NGHIEN CU...........................................................................38
I. LA CHON X LY NGUYEN LIEU..............................................................38
II. TACH CHIET HESPERIDIN. .................................................................................39
II.1. Phng phap va ieu kien chiet Hesperidin....................................................39
II.2. Tinh che Hesperidin.
.........................................................................................41
III. NGHIEN CU IEU KIEN TONG HP HESPERETIN............................43
III.1. ieu kien va quy trnh tong hp...................................................................43
III.2. Tinh che Hesperetin...............................................................................................45
IV. NGHIEN CU IEU KIEN TONG HP TRIACETYL HESPERETIN....46
IV.1. ieu kien va quy trnh tong hp....................................................................46
IV.2. Tinh che Triacetyl Hesperetin.............................................................................48
V. NHAN DANH SAN PHAM..................................................................................48
VI. XAC NH HOAT TNH KHANG NAM, KHANG KHUAN...................49
B. THC NGHIEM.........................................................................................................50
I. LA CHON X LY NGUYEN LIEU...............................................................50
II. TACH CHIET HESPERIDIN...................................................................................50
II.1. Phng phap va ieu kien chiet tach Hesperidin.
.................50
II.2. Tinh che Hesperidin................................................................................................50
III. NGHIEN CU IEU KIEN TONG HP HESPERETIN............................51
III.1. Khao sat qua trnh tong hp Hesperidin thanh Hesperetin...........................51
III.2. Tinh che Hesperetin. ............................................................................................52
IV. NGHIEN CU IEU KIEN TONG HP TRIACETYL HESPERETIN.....53
IV.1. Khao sat qua trnh tong hp Hesperetin thanh Triacetyl Hesperetin...........53
IV.2. Tinh che Triacetyl Hesperetin.............................................................................53
V. NHAN DANH SAN PHAM...................................................................................54
V.1. Nhan danh Hesperidin...........................................................................................54
V.1.1. Sac ky lp mong, o nhiet o nong chay...........................................54

Hoc vien Nguyen Th Bch Tram


Trang 115

Luan van thac s


V.1.2. Xac nh bang pho UVVis.......................................................................54
V.1.3. Xac nh bang pho IR ................................................................................54
V.1.4. Xac nh bang pho LC-MS.........................................................................55
V.2. Nhan danh Hesperetin. .......................................................................................55
V.2.1. Sac ky lp mong, o nhiet o nong chay.........................................55
V.2.2. Xac nh bang pho UV-Vis.......................................................................55
V.2.3. Xac nh bang pho IR.................................................................................55
V.2.4. Xac nh bang pho HPLC.........................................................................55
V.3. Nhan danh Triacetyl Hesperetin...........................................................................55
V.3.1. Sac ky lp mong, o nhiet o nong chay.........................................55
V.3.2. Xac nh bang pho UVVis.....................................................................55
V.3.3. Xac nh bang pho IR. ...............................................................................55
V.3.4. Xac nh bang pho HPLC.........................................................................56
V.3.5. Xac nh bang NMR...................................................................................56
VI. XAC NH HOAT TNH KHANG NAM, KHANG KHUAN...................57
VI.1. Cac loai vi khuan, nam th nghiem.............................................................57
VI.2. Moi trng th nghiem....................................................................................57
VI.3. Chat th nghiem................................................................................................58
VI.4. Tien hanh khao sat...........................................................................................58
I. TACH CHIET HESPERIDIN.....................................................................................60
I.1. Tach chiet Hesperidin...........................................................................................60
I.2. Tinh che Hesperidin.
..........................................................................................60
II. NGHIEN CU IEU KIEN TONG HP HESPERETIN.............................60
II.1. Khao sat qua trnh tong hp............................................................................60
II.1.1. Khao sat ve thi gian phan ng...........................................................61
II.1.2. Khao sat ve lng Methanol tham gia phan ng. ..................................62
II.1.3.Khao sat ve lng H2SO4 96% tham gia phan ng. ..............................63
II.2. Tinh che Hesperetin................................................................................................65
III. NGHIEN CU IEU KIEN TONG HP TRIACETYL HESPERETIN....66
III.1. Khao sat qua trnh tong hp Hesperetin thanh Triacetyl Hesperetin...........66
III.1.1. Khao sat ve thi gian phan ng...........................................................66
III.1.2. Khao sat ve lng (CH3CO)2O tham gia phan ng. ..........................68
III.1.3. Khao sat ve lng CH3COONa tham gia phan ng. ...........................69
III.2. Tinh che Triacetyl Hesperetin...............................................................................71
IV. NHAN DANH SAN PHAM.................................................................................72
IV.1 Nhan danh Hesperidin. .........................................................................................72
IV.1.1. Sac ky lp mong, T0nc. ........................................................................72
IV.1.2. Pho UV-Vis (Ethanol)....................................................................................72
IV.1.3.Pho IR..............................................................................................................72
IV.1.4. Pho LC MS : (xem phu luc 7,10,11,12)....................................................73
IV.2. Nhan danh Hesperetin. ........................................................................................74
IV.2.1. Sac ky lp mong, T0nc............................................................................74
IV.2.2. Pho UV-Vis (Methanol).................................................................................74
IV.2.3.Pho IR............................................................................................................75
IV.2.4. Pho HPLC.(phu luc 8)..................................................................................75
IV.3. Nhan danh Triacetyl Hesperetin. .........................................................................76

Hoc vien Nguyen Th Bch Tram


Trang 116

Luan van thac s


IV.3.1. Sac ky lp mong, T0nc..........................................................................76
IV.3.2. Pho UV-Vis (Methanol).................................................................................77
IV.3.3.Pho IR............................................................................................................77
IV.3.4.Pho HPLC. (Xem phu luc 9).........................................................................77
IV.3.5. Pho NMR. .............................................................................................77
V. XAC NH HOAT TNH KHANG NAM, KHANG KHUAN. (xem phu
luc 19)..................................................................................................................................81
TAI LIEU THAM KHAO...........................................................................................86

PHU LUC
BANG TOM TAT QUA TRNH HOC TAP, CONG TAC VA
HOAT ONG KHOA HOC KY THUAT

Hoc vien Nguyen Th Bch Tram


Trang 117

Luan van thac s

DANH MUC CAC PHU LUC

Hoc vien Nguyen Th Bch Tram


Trang 118

Luan van thac s


DANH MUC CAC BANG

Bang 1 : Ket qua khao sat bien oi Hesperidin thanh Hesperetin theo thi gian
phan ng...........................................................................................................................61
Bang 2: Ket qua khao sat bien oi Hesperidin thanh Hesperetin theo lng
Methanol tham gia phan ng............................................................................................62
Bang 3 : Ket qua khao sat bien oi Hesperidin thanh Hesperetin theo lng
H2SO4 96%..........................................................................................................................64
Bang 4 : Ket qua khao sat bien oi Hesperetin thanh Triacetyl Hesperetin theo
thi gian phan ng..........................................................................................................66
Bang 5 : Ket qua khao sat bien oi Hesperetin thanh Triacetyl Hesperetin theo
lng (CH3CO)2O tham gia phan ng...........................................................................68
Bang 6 : Ket qua khao sat bien oi Hesperetin thanh Triacetyl Hesperetin theo
lng CH3COONa tham gia phan ng..........................................................................69
Bang 7: 1H-NMR cua Triacetyl Hesperetin.....................................................................77
Bang 8: 13C-NMR cua Triacetyl Hesperetin...................................................................78
Bang 9 : Ket qua xac nh tnh khang nam khang khuan...................................81

DANH MUC CAC S O

Hoc vien Nguyen Th Bch Tram


Trang 119

Luan van thac s

DANH MUC CAC O TH

DANH MUC CAC HNH

Hnh 1: Cau truc hoa hoc cua Hesperidin....................................................................18


Hnh 2 : Cong thc cau tao cua Hesperetin ................................................................23
Hnh 3 : Cong thc cau tao cua Triacetyl Hesperetin..................................................26
Hnh 4 : He thong un hoi lu........................................................................................51

Hoc vien Nguyen Th Bch Tram


Trang 120

Luan van thac s


BO GIAO DUC VA
Cong hoa xa hoi chu
AO TAO
ngha Viet Nam
TRNG AI HOC CAN
oc Lap T Do Hanh
TH
Phuc
--------------------------------NHIEM VU LUAN VAN THAC S
Ho Ten hoc vien
: NGUYEN TH BCH TRAM
Sinh ngay
: 07 / 08 / 1979
Chuyen nganh : HOA HOC HU C.
I. TEN E TAI : Tach chiet Hesperidin, tong hp
Hesperetin, Triacetyl Hesperetin va xac nh
hoat tnh khang nam, khang khuan cua
chung
II. NHIEM VU VA NOI DUNG :
-

Tach chiet Hesperidin,


Tong hp Hesperetin,
Tong hp Triacetyl Hesperetin,
Xac nh hoat tnh khang nam, khang khuan
cua Hesperidin, Hesperetin va Triacetyl Hesperetin.

III. NGAY GIAO NHIEM VU


: 15 / 10 / 2004.
IV. NGAY HOAN THANH NHIEM VU
:
/ 11 / 2005.
V. HO TEN CAN BO HNG DAN CHNH : Ts. NGUYEN
CU KHOA.
VI. HO TEN CAN BO CHAM NHAN XET 1 :
VII. HO TEN CAN BO CHAM NHAN XET 2 :
CAN BO HNG DAN
BO NHAN XET 2

CAN BO NHAN XET 1

CAN

Noi dung va e cng luan van Thac S a c thong


qua hoi ong chuyen nganh.
Ngay thang nam
2005
PHONG QLKH SH

Hoc vien Nguyen Th Bch Tram


Trang 121

Luan van thac s


CHU
NGANH

NHIEM

CONG TRNH C HOAN THANH TAI


PHONG CONG NGHE HU C CAO PHAN T
VIEN CONG NGHE HOA HOC

CAN BO HNG DAN KHOA HOC :


Ts. NGUYEN CU KHOA.

CAN BO CHAM NHAN XET 1 :

CAN BO CHAM NHAN XET 2 :

Luan van c bao ve tai HOI ONG CHAM BAO VE


LUAN VAN THAC S TRNG AI HOC CAN TH.
Ngay
2005

Hoc vien Nguyen Th Bch Tram


Trang 122

thang

nam