Nghiên cứu hoạt tính kháng khuẩn, kháng oxi hóa, ức chế tyrosinase của cao ethanol chiết xuất từ cây huỳnh anh ( allamanda neriifolia)

TAP CHI PHAT TRIk KUM TAP 17, 30 T3 - 2014

Nghien ci:ru hoat tinh khang khugn,
khang oxi hoa, Crc the tyrosinase cua
cao ethanol chiet xuat ti' cay Hujmh
Anh (Allamanda neriifolia)
• Trin Pham Tue Hung
• Nguyen Thi M9 Hanh
• Quach NO Diem Phirang
Tru&ng Dai hoc Khoa hoc To, nhien, DHQG-HCM
nkin n,gify

29 (hang 07 kin; 2014, n125)2 clang 'Tay 16 dicing 01 friar)? 2015)

TOM TAT
ce
vai cac del ttrimg cOn lai. Dinh tinh
Cao ethanol cac 60 phen re, than trot,
mat
cac
hop
chit
thir
cap
trong
cao
ethanol
than leo, la dm cay Huynh anh (Allamanda
ter than leo cho they su' hien dien caa
neriifolia) ductc khdo sat kha nang khang
alkaloid, steroid, triterpenoid, phenol, tannin

the
tyrosinase.
khuan, khang oxy haa, CPC
va flavonoid. Phan tich clang flavonoid cho
Trong nhang be phen nay, cao ethanol ter
they meu c6 the c6 flavan hoac chalcone.
than leo the hien cac hopt tinh cao Met so
Tel' kh6a: Cfc the tyrosinase, Huynh Anh (Allamanda neriifolia), khang oxi h6a.
MD D AU
Nam da, sam den la cac dau hieu benh lY do
sir bieu hien vuat mire melanin a da. De khic
phuc cac trieu chi:mg nay, nguiri to thuang sir
dung cac hoa chat có kha nang irc the tyrosinase—
enzyme tham gia sinh tong hop melanin.Tuy
nhien, viec sir dung cac hoa chat nay co the gay
ra tac dung phu cho nguai ding; trong s6 do, mot
s6 'oat da bi cam sir dung dr nhieu nuac. Thay vao
do, nhiing hoat chat chiet xuAt tir dime lieu co
kha rang irc the tyrosinase dirge sir dung bar
hieu qua lam tring da va tinh an loan [11].
Cay Huynh Anh co ngu6n gOc Trung va Nam
My. Ben canh gia tri lam canh, nhieu loci trong
da- ductc
chi nhu A. carthartica, A. blanchetti
chUng minh co nhieu die tinh dune hoc khac
eing nhir tic the tyrosinase. Nghien ciru cua
Omonhinmin A va ding sir nim 2011 di chimg
minh kha Jiang cita A. cathartina trong chita trt
sot ret in vivo bing ca the kith thick hoat dOng

cUa cac he thOng chOng oxi h6a cua co the. Dich
chiet re, la va hoa da chrac thing minh co kha
nang khang su hinh thanh khOi u in vitro tren to
boo K562 leukemia... [3-5,7,8].
Melanin la nhirng hop chat mau, .dang
polyphenol, ton tai a da, long, t6c, mAt nguai,...
N6 gia vai tro quan trong trong can hang the oxi
h6a cua da bai kha nang bit g6c tir do. Melanin
duac sinh tong hap a melanocyte, bit dau tir ca
chat L-tyrosin vai su tham gia cda enzyme
tyrosinase. Do vay, irc the enzyme tyrosinase,
lam glom luong melanin tao thanh [6].
VAT LIEU VA PHOING PHAP
Nguyen lieu
Cay Huvnh Anh dirge thu hal tai Quan Thu
Dire, Thanh phi-% HO Chi Minh va cluac dinh danh
tai BO mon Sinh thai va. Sinh hoc tien hoa,
Truing Dai hoc Khoa hoc Tu nhien, DHQGTrang 61


Science & Technology Development, Vol 17, No.T3- 2014

HCM. Ben be phan ctia cay: re-, than true, than
leo va la duqc thu hal, say kho, xay nho.
Phtron phip
Dietz the cao ethanol
Cao ethanol da cac be phin dune, dieu the
bang phtrang phap ngam trong 24 gi6. Sau khi
ngam va chiet 3 lin, cac mau duqc co quay thu
cao tit&

Khao sat hogt tinh khang khudn bang do during
kinh yang vo khudn
Cac chimg khuan sir dung: Staphylococcus
aureus K, Staphylococcus aureus, Bacillus
subtilis, Shigella flexneri, Escherichia coil,
Salmonella spp, Pseudomonas spp dtr6c hoot
h6a va trai tren mai trung LB.
Cao ethanol 4 be phin cita cay duqc pha
loang trong ethanol tuyet dOi va dat len &a giay
5mm vai nOng de 500 gg/cria. Kanamycin ding
de 30 µg/dia lam chimg dirong, ethanol 5 µl/dia
lam chimg am.
Khcio sat Ming hit khzi theo Yen va Duh (1993)
Cao ethanol 4 be phan cita cay va vitamin E
(cluing dtrcmg) &Km pha bang trong ethanol
tuyet ClOi voi nang de 500.tg/ml. Ethanol tuyet
d6i la cluing am. Hut Ian lust 1 ml cac dung dich
ten vao Ong nghiem nang biet, b6 sung 2,5m1
dung dich dem Na2(PO4)30,2M - pH 6,6, IL deu,
them 2,5 ml K3(Fe(CN)6) 1% va u a 50 °C, 20
phin. Them 2,5 ml TCA 10% va ly tam 7000
Ong trong 2 phirt.Thu 1 ml dich nOi, them 2m1
ntr6c cat, 0,5 ml dung dich FeCI3 1%. San pham
thu (lucre do de hap thu a 700 nm.
Khao sat kha nang bdt gdc to do DPPH
Cao ethanol 4 be phin cua cay duos pha
loang trong ethanol tuyet d6i thanh day nOng
khac nhau. Vitamin E la chimg ducmg, ethanol
tuyet dei la chting am. Hut Ian loot 0,5m1 cac
dung dich tren vao tirng Ong nghiem, them 3m1

ethanol tuyet d6i, 0,5 ml dung dich DPPH 0,6
mM duqc pha trong ethanol tuyet dOi. Lac deu
hem imp, u tai 36 phut a nhiet de ph6ng va do de
Trang 62

hap thu a bloc song 517 nm. Phan tram bat gee
ti do DPPH duqc 'the djnh bang ding thire:
0/0
[(A thong am - A blank) - (A thu nghiem - A blank)] x 100
IC
chung am - A blank)
(1)

Lutz ch9n dung mai pha cao va khao sat kha
nang tic chef tyrosinase
70 gl dung dich cao ethanol than leo pha
loang trong ethanol tuyet ai, DMSO 1%, DMSO
100% thanh day nOng de khac nhau duqc nap len
dia 96 gieng. Mau thing ant khong chira chit thir
nghiem. Them 30 pi dung dich tyrosinase nOng
de 333 U/m1 va 0 a nhiet de phong 10 phut.
Them 110 gl L-tyrosine 2 mM va tiep tic 0
trong 30 Olin. Tien hanh do de hap thu a btrac
song 490 nm. Kojic acid ducfc diing lam chimg
during. Cac miu di kern vai mk blank kh8ng
chira tyrosinase. Phan tram irc the tyrosinase
duqc xtic djnh bang cling thirc (1).
Tien hanh so sanh kha nang uc the
tyrosinase cua cao ethanol 4 be phin cda cay
ming a 550 µg/ml bang each lap lai thi nghiem

nhu ten.
Dinh tinh cac h9p chdt thti cap tren matt cao
ethanol than leo
Cao than leo pha bang trong ethanol tuyet del
nOng a 1 mg/ml va djnh tinh sir hien dien cac hop
chat thir cap bang thuOc thin khac nhau: alkaloid
(Wagner), triterpenoid - steroid (Lieberman
Burchard, Salkowski), saponin (HCI 0,1N, NaOH
0,IN), phenol (FeC13 5%), tannin (gelatin man),
flavonoid (chi acetat, H2SO4 dam dac, NaOH 1%,
Shinoda). Cac mau thin di kern v6i Mau chimg am
chi c6 dung mei va thu6c thu [I].
Phurmg phap phan tick va tie li so lieu
Cac se lieu dtruc xir 19 bang phan mem SAS
9.1.3 Service Park 4. Si khac biet c6 9 nghia ye
mat th6ng ke a mac P (p-value) < 0,05. Cac sO
lieu dupe With bay dm% dang trung binh ± de
, lech chuin.


TAP CHI PHAT TRIA KH&CN, TAP 17, Si T3 - 2014

Be
KET QUA VA THAO LUAN
Cao ethanol cac be phan re, than true, than
leo, la dm cay Huynh anh c6 hien suet chiet cao
len lust 10,23; 4,63; 4,47; 34,12 (%) tinh tren
trong luting kho. So voi cac be phan con lot; la co
chira nhieu hqp chat hem tan trong ethanol nhat.
Khoo eat hoot tinh khang khuin cua

cao
chat

O Bang 1 ket qua khang khuan caa
kanamycin chi:mg mink thi nghiem hoot Ong Veit
vi min chimg am ethanol chimp mirth hoot tinh
khang khuan cea cac loot cao hoan toan do cac
hcrp chit co trong cay.
6 ming dij 500 1.1g/dia, cao than leo the hien
hoot tinh khang khan monh dei vied S. aureus —

vi khan gay cac berth viem da, co the troy thanh
nhan 16 Op pan quan trong trong thanh phan
my phi= &Ong da. Cao than trvc c6 hoot tinh
khang monh dei vol Samonella spp, S. aureus.
Cao re kiting monh voi E. colt, Samonella spp, S.
aureus. So voi nghien tiro cna Mohammad
Rashedul Islam (2010), cac phan don khac nhau
tircao methanol la caa 1 loci day hujrnh khic
(Allamanda carthartica) deu the hien kha nang
khang khan thap cho 2 chtIng S. aureus va E.
colt. Trong nghien ciru cua Swjanja Pulipati va
tong sir (2012) en v&i hoa cua cay Allamanda
carthartica, khi ly trich bang ethanol, voi ming
de 500 µg/die cho hoot tinh khang khan trung
birth yeti S. aureus va E. colt [10].

Bing 1. Ket qua Idiao sat hoot tinh khang khuan tir cao ethanol 4 be phan cea cay (don vi tinh: mm)
Chung vi khan

La

Thin leo

Than tine

Re

Kanamycin

Ethanol

S. aureus K

6,00:1-1,00c

7,67±1.15c

7,33=0,58,

12.33±2.08b

19,67*1,53a

6,67±0,58c

S. aureus

13.67=1,53b

18,33±2,08°

17:67+1,51°

16,31+1436

17,33±3,051

6.67±1,15c

Shigella flemeri

5,67±0.58c

7,33±0.58,

5,67±0,58c

12,00=1,006

18,67±2,894

6,33*0,5Ss

B. subrilis

7,00:4,00

6,00±1,006

5,67=0.58'

7,33±I, l 5b

21.00±1,00'

6,67*l,53b

13,33±.2,086

14,33±0,586

14,67±0.586

18,6711,15°

19,67±0,581

6,67±1,53c

8,33±2,08c

8,67±-1.53,

13,67±4.516

14.67=1.53'

21.67±1.53,

6,67±0,58c

11,33=2,08,

10,67=2.08C

19,670.58"

16,33±3,21'

21.33=1.53'

6,33=0.58d

khdo sat '

E.

coil

Pseudomonas
spp.
Samonella spp.

(a, b, c thir phan hang)
Paulo sat nang lire khir cast

cao chat

Bang 2. Ket qua khao sat nang lirc khir caa cac cao chiet

OD700

Vitamin E

Re

Than trvc

Than leo

Là

Ethanol

0,72+0,02'

0,234,02'

0,17±0,00d

0,52±0,056

0,19+0,00'd

0,10+0,02'

Trang 63


Science & Technology Development, Vol 17, No.T3- 2014

Hinh Ket qua khao sat Ming luc kb./ cna cac cao chiet
(Y1- vitamin E, Y2- re, Y3- than true, Y4- than leo, Y5- la, Y6 chUng am)
Dua vao Bang 2, a 4 loai cao ethanol cila
cac bd phan re, than true, than leo, lab nOng dd
500 lig/m1 deu the hien nang luc khi sit. Tit a
cac min thi nghiem deu co gia tri dd hap thu xep
hang cao hcrn mau chirng am ethanol. Cao than
leo co nang lut khir math nhat, gat bing 3/4 men
chirng duong vitamin E.

Khao sit kha nang bit gric tty do DPPH cua
die cao chiet
Tir &rang cong the hien tuang quan tuyen
tinh gifra ndng dd meu thi.1 va gia tri % bit cac
g6c to do DPPH (Hinh 2) not suy duot gia tri
ICso irc the DPPH ciia cac men cao thi nghiem.

100,00

100,00

x
4 so,00

80,00
Op

60 00
OS

6

60,00

'

40,00

40,00

1 20,00

if

0,00

20,00
0,00

7,81 15 63 31,25 62,50 125,00250,00

CP ,,pro

4"'

— Dl

Wing da uglml

CPCP D3

en

Ning do ughnl
—D5
Huth 2. Phin trim bit g6c to do DPPH dm vitamin E va cac cao ethanol tit 4 bd phan cay Huynli anh 6 cac ding
dd khac nhau (Dl- vitamin E, D2- re, D3- than true, D4- than leo, D5- la)
Bang 3. Gia tri 1050 irc the DPPH cua cac cao chiet to 4 bd phan cay Hujmh Anh

IC50

Vitamin E

R6

Thin Ulm

Thin leo

La

62,04±17,970

713,44±188,896

1397,24± 263,800

100,65±2,830

936,8633,696

Dua vao Bang 3, cao than leo co gia tri IC50
thAp that so viii 3 bd phan con lai, dot 100,65
µg/ml. So WU gia trj 1050 ctla vitamin E la 62,04
ttg/ml, cao than leo the hien kha nang bit g6c to
do rat match. Ket qua nay cang Wang &tong v&i
nghien ccru dtroc trinh bay trong boo cao oda
Amjad Hameed va tong su nom 2014 mla loci A.
cathartica [21
Trang 64

Lira chon dung mai pha cao va khao sit kha
nang tic ch6 tyrosinase
Sit dung ethanol tuyet ddi va DMSO 100%,
gia trj % itc the tyrosinase ctla mitt khOng tuong
quan ding dd chat thit, nghiem. Ngtroc lai, khi sir
dung DMSO 1% pha loang cao, gia tri % itc the
tang theo ding dd chAt thit nghiem (Bang 4). '


TAP CHI PHAT TRIEN DUN, TAP 17, S0 T3 - 2014
Bang 4. Gia trt irc the tyrosinase cue cac cao than leo pha trong cac dung m8i khac 'Mau
% tic the
dm kojic acid

Nang (10
kojic acid

Nang da.

mazu TN
(jig/m1)

% tic the
ink TN pha
trong DMSO
100%

% trc the
mL TN pha
trong ethanol
100%

% tic the
Mitt TN pha
trong DMSO
1%

500

97,08

2000

-344,53

-1,46

87,07

250

96,35

1000

-102,19

18,25

74,14

125

96,35

500

-32,12

29,93

50,13

62,50

86,13

250

18,25

18,25

35,09

31,25

75,18

125

57,66

9,49

27,70

15,63

54,74

62,50

64,23

5,84

19,79

7,81

4,38

31,25

65,69

13,87

17,68

3,91

1,46

15,63

62,77

21,90

14,78

Nguyen nhan c6 the do DMSO va ethanol c6
kha nang lam mat tinh en dinh cau trim protein,
Anh huang den hoot tinh ctYa tyrosinase trong thi
nghiem. Theo Tjemberg va cong su (2009), ming
do DMSO > 5% c6 kha nAng anh hirang den
enzyme vii st 'Nem cac tap chat trong DMSO

tang nhu ethanol cam den tai he qua tucmg tu.
Nai suy tit Wang quan tuyen tinh gifta nOng
de, mei' thtY va gia tri % irc the tyrosinase (Bing
4) cdo &rem gia tri IC50 irc the tyrosinase cite mL
cao than leo (Ma tan trong DMSO 1%) la 537,63
µg/ml va mau chimg duong kojic acid dat 9,86

nOng 65 550

gen bang gia tri IC50

cda cao than leo, cac mit' than true, re, la dew c6

gia tri % irc the thap han men cao than leo. So
yeti cite nghien cim khac, trong ding 1 chi
Allarnanda thi meu than leo dtrac thtt nghiem
(Allarnanda neriifolia) c6 hoot tinh chap han
Allamanda carthartica ninmg cao han
Allamanda blanchetti. Cao than leo ming the hien
hoot tinh irc the tyrosinase cao han cil nghe
Curcuma longa vai IC50 785,10 lig/m1 ducir clan
gian sir dung him ma WO, tri them den [4, 5, 9].

Bing 5. Phan tram irc the tyrosinase cda cao chiet tir 4 ba phan cay Huynh Anh d nOng dO 550µg/ml

% Tic the

Re

Than true

Than leo

La

27,671:0,441'

40,60±1,85'b

59,61+7,08'

15,40±4,25`

(a,b,c thee hr pilau hang)
Binh tinh cac hqp chit thfr cap tren mau cao thin leo cay Huynh Anh
• Bing 6. Ket qua dinh tinh cac nhom chat hoa tux tren matt cao than leo
Alkaloid

Triterpenoid

Steroid

Saponin

Phenol

Tannin

-+

+

+

-

+

+

Flavonoid
+
Trang 65


Science & Technology Development, Vol 17, No.T1 2014

(")

111%

Triterpenoid iabot

Flavonoid

Hinh 3. Ket qua djnh tinh cac nhom chat him hoc tren mau cao than leo
Ket qua trinh bay trong Bang 6 va Hinh 3
ding Mang Bong voti nghien dru cda Elita Scio

va cOng str, khi sang loc cac 'Thom chile tren mAu

la cao Allamanda cathartica.

Bang 7. Bang phan tich thanh phAn flavonoid bang cac phan irng mau

Thi nghiem

Nhom chat dir doin

Hien timing
Flavane

Chi acetate

Tda vang

+

Shinoda

Vang nhat

+

H2SO4

Cam, do

NaOH

Cam, do

Trang 66

Flavanone

Flavone

Flavonol

Flavanonol

Chalcone

+

+

+

+

+
+

+

+

+

+


TAP CHI PHAT TRIbl Ith&CH, TAP 17, 30 T3 - 2014

ITInh 4. Ket qua cac thi nghiem phamtich thanh phan flavonoid
Bang cac phial img vOi cac thutic tit* co the dir
(loan dugs clang flavonoid chinh trong cao than leo.
Khi phan ung voi chi acetate, hau het cac nhOm deu
co kha ning tao phirc Ma. Trong phan Ong Shinoda,
mau c6 mau yang nhat (am tinh) chimg to cac
flavonoid clang flavane hoac chalcone. Khi phin
fmg yeti H2SO4, NaOH, sin pham tau cam, do
chimg to min thin co flavanone, flavonol va
chalcone. Tuy nhiert, nen trong min tit* c6
flavanon, flavonol thi phan img Shinoda phai c6
mau dO, cam. Vay nen trong min khong co
flavanone, flavonol. Mat khac flavan 1:4 bien tinh

tao mau cam, do kin tic dung veri acid hay kiem
manh. Vi vay, flavonoid trong cao thin leo th the la
chalcone va flavane [I].
KET LU4Isi
Cao thin leo Huy_ rth Anh co hoot tinh khang
khan (S. aureus), khang oxy h6a, frc the
tyrosinase cao nhit. Trong cao thin leo co:
alkaloid, triterpenoid, steroid, phenol, tannin,
flavonoid. Flavonoid c6 the 6 clang flavane hoac
chalcone.

Trang 67


Science & Technology Development, Vol 17, No.T3- 2014

Studying antibacterial, antioxidantand
tyrosinase inhibition activities of golden
trumpet (Allamanda neriifolia)
• Tran Pham Tue Hung
• Nguyen Thi My Hanh
• Quach Ngo Diem Phuong
University of Science, VNU-HCM

ABSTRACT
Golden Trumpet (Allamanda neriifolia)
Phytochemical identification of buds'extract
was investigated through antibacterial,
indicated the presence of alkaloids, steroids,
antioxidant, anti-tyrosinase activities. Ethanol
triterpenoids, phenols, tannins, flavonoids.
extracts of plant'parts: leaves, stems, roots,
Flavonoids possibly are flavanes or
buds were used, which resulted in the most
chalcones.
highlighted qualification of buds.
Key words: Tyrosinase inhibition, Golden Trumpet (Allamanda neriifolia),antioxidant.

TAI LIEU TRAM KHAO
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Isolation, identification and tyrosinase
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[5]. I. Batubara, L.K. Darusman, T. Mitsunaga,

G. Prota, Progress in the chemistry of
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Fonseca , E.C. Silva, D. Silveira , P.O.
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communication: Chemical composition,
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antioxidant assessment of two antimalarial
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T413 CHI PHAT TRIEN ITH&CM, TAP 17, 30 T3 - 2014
with potent tyrosinase inhibitor• activity,
Plos one, 7(11), 48589 (2012).
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(2012).

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Hongbo, Z.D.Q.H.I. Maibach., Human in
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7 rang 6')