Tiiu ban: cdc chat co Iwgl linh sinh hqc ISBN: 978-604-913-012-0<br />
<br />
<br />
<br />
NGHIEN c t r u THANH PHAN VA DlTOC TINH NOC BO CAP<br />
HETEROMETRUS LAOTICUS PHAN BO O TAY NINH<br />
<br />
Vo Do Minh Hoang', Pham Nguyen Dong Yen\ Nguyin Thi Phuong Khue ,<br />
<br />
Vo Phung Nguyen^ Hoang Ngoc Anh'<br />
<br />
'Vien Khoa hoc Vat lieu img dung, Vien KH&CNVN, 1-Mac DTnh Chi, Q.I, Tp. HCM<br />
^Dai hoc Y-Dugc, Tp.HCM<br />
Email: hnanh(a),vasl-hcm. ac. vn<br />
Tom tat:<br />
Bd cgp den Heterometrus laoticus (hg Scorpionldae) phdn bd nhiiu & cdc viing<br />
Tdy Nam Bd. Trong ddn gian bd cgp ngdm rugu dugc su dung rdng rdi di ldm<br />
thudc gidm dau co, dau thdn kinh tog, dau xuong... 'Tuy dugc ung dimg nhu vdy<br />
Irong ddn gian, nhung nge bd cgp H.laoticus cho din nay vdn chua dugc nghiin<br />
cuu. Tru&c linh hinh ddy chimg Idi liin hdnh nghien ciru ndy.<br />
Nge dugc thu lu bd cgp H.laoticus phdn bd & Tdy Ninh. Ldm sdc ky lge gel trin<br />
cdt Bio-Gel P 10 chiing Idi dd tdch nge bd cgp ldm 10 phdn dogn. Cdc phdn dogn<br />
ndy cd nhirng tinh chdt hod hgc vd sinh hoc khdc nhau. Thir ddc dnh cua cdc phdn<br />
dogn len chugt cho thdy cd hai phdn dogn cd ddc tinh. Nhirng phdn dogn ddc cd<br />
khdi lugng phdn tu ndm Irong viing 3-8 kDa vd chung Id cdc neurotoxin. Nhung<br />
nghien cuu dugc tinh cua nge bd cgp cho thdy nd cd tinh giam, dau vd khdng<br />
viem. Ddc linh cdp ciia nge bd cgp dugc xdc dinh la 170 ± 9,95 mg/kg chugt (tiem<br />
du&i da). Ddc linh ciia nge bd cgp dugc xep vdo ddc logi ba. Nge bd cgp cd khdng<br />
nhirng cd linh gidm dau trung uang vd gidm dau ngogi biin md cdn cd tinh<br />
khdng viem.<br />
Abstract:<br />
The black scorpion Heterometrus laoticus, family Scorpionldae, are wide<br />
distributed in the South - West Vietnam. In the traditional medicine the alcohol<br />
extract of these scorpions is used as analgesic for muscle and bone pain.<br />
However, the venom of Vietnamese H. laoticus has not been studied untill now, so<br />
we carried out this work.<br />
Venom was collected from H.laoticus scorpions distribuding in the Tdy Ninh<br />
province. The crude venom of H.laoticus scorpions was separated into ten<br />
fractions by column chromatogaphy on Bio-Gel P 10. These fractions have<br />
different chemical and biological properties. Tests on mice showed that two of<br />
these fractions are toxic. The toxic fractions have molecular weight in range of 3-<br />
8 kDa, and they are scorpion neurotoxins. The pharmacology study showed the<br />
scorpion venom have analgesic and anti-inflammatory acdvitle. The LD50 of<br />
venom was 170 ± 9.95 mg/kg mouse (s.c). Toxicity of scorpion venom was<br />
classified ito the third class toxicity. The scorpion venom showed not only cenral<br />
and peripheral analgesic effect in acetic acid induced writhing lest and tail<br />
immersion test, but olso anti-inflammatory effect in carrageenin induced<br />
inflammation test.<br />
<br />
150<br />
Hoi nghi Khoa hqc ky niem 35 ndm I 'ien Khoa hqc vd Cong nghe Viet Nam - Hd Noil 0/2010<br />
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<br />
I. G i d l THIEU CHUNG<br />
Bd cap Heterometrus laoticus phan bd nhilu d cac vimg Tay Ninh. Rugu ngam bd c^p<br />
nay dugc su dung trong dan gian lam thudc xoa bdp giam dau co; thudc sac bd cap dimg dieu<br />
tri ddng kinh, co giat [1] nhung ddc tinh va tac dung dugc ly ciia noc bd cap chua duoc<br />
nghien cim nhilu [2,3], ciing nhu chua dugc khai thac de nghien ciiu rdng rai lam ngudn<br />
nguyen lieu trong Y dugc. Nhiing nghien cim ciia chiing tdi gan day cho thay noc bd cap An<br />
Giang cd tac dung khang viem, giam dau [9, 11]. Ve co ban binh thai sinh hoc thi loai bd cap<br />
d An Giang va Tay Ninh la ciing mot loai la Bd cap Heterometrus laoticus. Tuy vay tac dung<br />
dugc ly ciia chiing khdng hoan toan gidng nhau. Mot sd nghien ciiru noc ran cho thay thanh<br />
phan cac toxin trong noc khdng nhirng chi phu thudc vao loai ran ma cdn phu thudc vao vi tri<br />
phan bd ciia chiing [4, 5]. Do dd, trong bai bao nay chung tdi trinh bay nghien ciiu thanh phan<br />
va dugc tinh noc bd cap Heterometrus laoticus phan bd tai Tay Ninh tao ngudn nguyen lieu<br />
dimg trong Y dugc.<br />
<br />
II. NGUYEN LIEU VA PHLTONG PHAP NGHIEN ClTU<br />
1. Nguyin lieu<br />
a. Nge bd cgp<br />
Noc thu tir bo cap H. laoticus trudng thanh bat d viing Tay Ninh va nudi trong phdng<br />
thi nghiem ciia chiing tdi. Thirc an cua bd cap la chau chau va de. Thu noc bang phuong phap<br />
xung dien tai ddt thir 5 hoae thii 6 ciia dudi bd cap. Noc dugc lam khd trong binh hiit am cd<br />
chiia CaCli khan eho den kbi cd khdi Iugng khdng ddi, sau dd thu va giii d nhiet do -20°C cho<br />
den khi diing.<br />
b. Thii thu nghiim<br />
Chugt nhat trang gidng DDY, ca hai gidi, trong lugng tir 18-25 g do Vien Pasteur<br />
Thanh phd Hd Chi Minh cung cap. Chuot mua ve dugc dn dinh trong hai ngay trong phdng<br />
thi nghiem Dugc Iy Dai hoc Y Dugc TP.HCM trudc khi tien thir nghiem. Thdi gian thir<br />
nghiem tir 8-15 gid.<br />
2. Phirong phdp nghiin ciru<br />
a. Phuong phdp tdch cdc thdnh phdn ciia nge bd cgp H. laoticus phdn bd & vimg Tdy<br />
Ninh<br />
Noc bd cap Tay Ninh dugc phan tich bang phuong phap loc qua gel bang may FPLC<br />
(Bio-rad, My) d nhiet do phdng tren cot vdi Bio-Gel P 10 (1,5 x 100 cm). Cot da can bang<br />
trudc trong dem ammonium axetat 20 mM (pH 4,7), lugng noc mdi lan chay la 180-200 mg,<br />
tdc do rira cot 0,16 ml/phiit, the tich mdi dng thu 4 ml.<br />
Sir cd mat ciia protein trong cac phan doan sac ky dugc xac dinh theo mat do quang tai<br />
bude sdng 280 nm.<br />
Ddc tinh cua cac phan doan tach ra dugc thir len chugt nhat trang DDY ca hai gidng<br />
(18-20 g) bang each tiem dudi da, tac dung cua cac phan doan len chugt dugc quan sat trong<br />
24 gid. Lugng protein tiem vao mdi con chuot la 0,2 mg.<br />
Dien di dugc tien hanh tren gel polyachrylamit 14%) theo Laemmli [10].<br />
b. Nghien ciru ddc tinh cdp du&ng tiim du&i da tren chugt nhdt trdng [14]<br />
1. Nghien ciru so khdi: Khdi ddu tir lilu cao nhdt cd thi qua dudng tiem. Xac dinh lilu<br />
LDo (lilu tdi da khdng gay chit) va lilu LDioo (lilu tdi thilu gay chit 100%o).<br />
1. Nghien cuu xdc dinh: Chuot dugc chia lam 6 Id, moi Id it nhdt 6 con, cho sir dung<br />
thudc d cac lieu trong kboang LDQ va LDioo , chia theo cdp sd. C> nhimg lilu gdn LD50 tang<br />
sd lugng chuot de sir do ludng dugc chinh xac hon. Quan sat chuot trong 24 gid sau khi dung<br />
thudc, ghi nhan cac phan img xay ra tren chuot va sd Iugng sdng, chit d moi Id. Theo ddi<br />
chuot cdn sdng sau 14 ngay kl tir ngay diing thudc. Giai phdu dai thi nhimg chuot cdn sdng<br />
151<br />
Tieu ban: cdc clidt co Iwgl tinh sinh hqc ISBN: 978-604-913-012-0<br />
<br />
<br />
va nhiing chuot chit sau khi cho udng. LD50 dugc tinh theo phuong phap Miller - Tainter va<br />
Karber-Behrens.<br />
Phuans vhdp Miller - Tainter<br />
Xac dinh LD50 tren ^idy logarit- xac sudt. Ve dudng bieu diln ti le % chuot chet theo<br />
lilu tren gidy logarit. Doc ket qua LD50 = m tren true hoanh va sau dd xac dinli do lech chuan<br />
E. Tir dd thi, ta suy ra m; probit 6,0 (84% chit); probit 4,0 (16% chit)<br />
2S<br />
LD50 = m ± E vdi £ = ,<br />
dlN'<br />
2S: Sir gia tang lilu cdn thilt dk gia tang kit qua Ien them 2 probit<br />
N': Tdng sd thu vat dimg d cac lilu tuong img vdi cac phan suat tir vong d trong<br />
khoang giira probit 3,5 va probit 6,5.<br />
Phuons phdp Karber-Behrens<br />
<br />
Y^ab<br />
LD,, = DJ<br />
Trong dd<br />
Df: Lilu toi thilu lam chet tat ca thii vat.<br />
a: Chi sd trung binh ciia sd thii vat chet d bai lieu ke tiep.<br />
b: Hieu sd giira bai lieu ke tiep.<br />
n: Sd thii vat diing d moi lilu hoae sd trung binh ciia nhiing tri sd tren (trudng hgp sd<br />
thii vat thay ddi d mdi lieu).<br />
c. Nghien ciru tdc ddng dugc ly<br />
Lilu nghien cim tac dung dugc ly dugc suy ra bang each lay 1/20 - 1/10 gia tri LD50.<br />
Chuot dugc chia thanh 4 Id, moi Id la 8-10 chuot. (2 Id thir d hai lieu 1/20 va 1/10 ciia gia tri<br />
LD50, 1 Id chimg va 1 Id ddi chiing), rieng nghien ciiu tac dung khang viem cdn them Id trang<br />
(tiem dudi da dung mdi pha thudc ddi chirng)<br />
d. Nghiin ciru tdc ddng gidm dau trung uong theo phuang phdp nhiing dudi chugt<br />
[15].<br />
Chuot dugc cd dinh vao ldng vdi dudi dugc tha tu do ra ngoai. Nhiing ngap dudi chuot<br />
khdng qua 5 cm trong binh nude d nhiet do dn dinh 50 ± 0,5 °C. Tiem thdi giat manh dudi<br />
chuot tinh tir luc nhiing dudi chuot vao nude den khi chuot giat manh dudi ra khdi nude, dugc<br />
ghi nhan trudc khi cho diing thudc va vao cac thdi diem 30, 60, 90, 120 phut sau kbi cho tiem<br />
thudc. Lau khd dudi chuot sau mdi lan do, neu chuot khdng phan iing sau 10 giay thi nhac<br />
chuot ra de tranh bdng dudi cho chuot. So sanh tiem thdi giat manh dudi chuot giira cac Id<br />
kbac nhau.<br />
e. Nghien ciru tdc ddng gidm dau ngogi hien iheo md hinh gdy dau qudn [15,16].<br />
Sau khi cho chuot sir dung thudc nghien ciiu 30 phiit, tiem phuc md lan lugt tirng<br />
chuot vdi 10 mL/kg acid acetic 0,7 %. Sd lan dau quan ciia chuot dugc quan sat va ghi nhan<br />
trong 5 phiit d cac thdi diem 5-10, 20-25 va 35-40 phut sau khi tiem acid acetic. Chuot dugc<br />
cho la dau quan khi quan bung va dudi it nhat mot chan sau. So sanh sd lan dau quan ciia<br />
chuot giiia cac Id tai cac thdi diem vdi nhau.<br />
/. Nghien ciru ldc ddng khdng viem iheo md hinh gdy phii chdn chudi bdng<br />
carrageenin [17].<br />
Carrageenin dugc cung cap bdi Sigma Alrich. dugc diing lam tac nhan gay viem. Do<br />
do phii chan chuot bang Plethymometer model 7140, hang Ugo Basile.<br />
152<br />
Hoi nghi Khoa hqc k\- niem 35nam 'ien Khoa hqc vd Cong nghe Viet Nam - Hd Not 10/2010<br />
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<br />
Chuot dugc gay viem bdng each tiem vao gan ban chan 0,025mL dung dich<br />
carrageenin 1 % pha trong dung dich sinh ly. Do the tich chan chuot 3 gid sau khi gay viem.<br />
Cac chuot cd the tich chan sung phii tren 50 % so vdi binh thudng dugc lira chon cho nghien<br />
cim va chia ngau nhien thanh cac Id va lan lugt tiem dudi da cac dung dich: Ld chiing: dung<br />
dich sinh ly 0,9 %; Ld ddi chirng: dung dich Ketoprofen (Profenid ®), 5 mg/kg/ benzyl<br />
alcohol 2,5 %; Ld trang: dung dich benzyl alcohol 2,5 %; Ld thir thudc dung dich noc bd cap<br />
d hai lieu khae nhau.<br />
Theo ddi the tich sung phii ciia chan chuot sau 3 gid gay viem va sau kbi tiem thudc<br />
30 phut mdi ngay vao mot gid nhat dinh trong 6 ngay lien tiep. Do tang the tich chan ciia timg<br />
chuot cdn goi la do sung phii, dugc tinh theo cdng thirc:<br />
V,-Vo<br />
AV%o = xlOO<br />
Vo<br />
VQ: The tieh chan chuot trudc khi gay viem (do 1/100 mL).<br />
Vt: The tich chan chuot sau khi gay viem (do I/lOO mL).<br />
g. Thdng ki phdn tich<br />
DO" lieu dugc trinh bay dudi dang sd trung binh ± SE. Sir kbac biet giira cac nhdm<br />
dugc phan tich bang phuong phap Kruskal - Wallis sau dd la Mann - Whitney test vdi phdn<br />
mem vi tinh Minitab 14.0. P < 0,05 dugc cho la cd y nghTa thdng ke.<br />
III. KET QUA VA BIEN LUAN<br />
1. Tdch cdc phdn doan cua nge bd cap H.laoticus phdn bo & Tdy Ninh<br />
Ket qua phan tich noc bd cap tren cot vdi Bio-Gel P 10 cho thay noc tach ra lam 10<br />
phan doan vdi khdi lugng phan tir khae nhau (hinh 1). Thir ddc tinh cac phan doan tach ra cho<br />
thay cd hai phan doan 6 va 7 cd doe tinh.<br />
<br />
<br />
<br />
<br />
.. i<br />
<br />
<br />
<br />
<br />
100 200 300 400 500 70O 80(1<br />
Time [minutesl<br />
<br />
<br />
<br />
<br />
Hinh 1: Phdn tich nge bd cap H.laoticus Tdy Ninh trin cot Bio-Gel P 10<br />
<br />
2. Khdo sdt ddc tinh cda cdc phdn doan tdch ra td nge bd cap Tdy Ninh<br />
D I khao sat tinh chat cua noc bd cap chiing tdi da thir ddc tinh cua 10 phan doan da tach<br />
ra tren chuot. Ket qua cho thay cd hai phan doan (6 va 7) cd ddc tinh cdn tam phan doan khae<br />
(1, 2, 3. 4. 5. 8, 9 va 10) khdng cd doe tinh. Sau khi tiem dudi da hai phan doan 6 va 7 vao<br />
chugt, chupt bj mat thang bang, co giat va chit trong khoang 1-2 gid. Cac phan doan nay tac<br />
ddng len he than kinh ciia chuot. nhu vay chiing chira cac neurotoxin. Kit qua thir ddc tinh len<br />
chuot ciia cac phan doan dugc trinh bay trong bang 1.<br />
<br />
153<br />
Tieu ban: cdc clidl co hogi linh sinh hqc ISBN: 978-604-913-012-0<br />
<br />
<br />
Bdng 1: Thu ddc tinh ciia cdc phdn dogn tdch ra tir nge bd cgp H. laoticus Tdy Ninh<br />
<br />
So phan Quan sdt trin chuot sau khi tiim cdc phdn doan tdch ra Nhdn xet<br />
doan<br />
I Chuot xii ldng, gai chan sau dd binh thudng Khdng doe<br />
2 Chuot xii ldng, gai chan, ngii, sau dd binh thudng Khdng ddc<br />
3 Chuot xii ldng, gai chan, ngii sau dd binh thudng Khdng doe<br />
4 Chuot xii Idng, gai chan, ngii sau dd binh thudng Khdng ddc<br />
5 Chuot xii Idng, gai chan, ngii sau dd inh thudng Khdng doe<br />
6 Chuot mdt thang bang, co giat va chet sau 2 gid Doe gay chet<br />
7 Chuot mat thang bang, co giat va chet sau 1 gid Doe gay chet<br />
8 Gai mieng, gai chan sau dd binh thudng Khdng doe<br />
9 Gai chan sau dd binh thudng Khdng ddc<br />
10 Gai chan sau dd binh thudng Khdng doe<br />
Nude cat Chuot boat ddng binh thudng Khdng doe<br />
Tiep theo de khao sat thanh phan protein eua cac phan doan tach ra, chiing tdi da tien<br />
hanh dien di cac phan doan tir 2 den 7. Ket qua dien di {hinh 2) cho tbay cac phan doan ddc la<br />
cac phan doan 6 va 7. Cac phan doan dd chiia cac protein cd khdi lugng phan tir nam trong<br />
viing 3-8 kDa tuong duong vdi khdi lugng phan tir ciia cac neurotoxin noc bd cap, va nhu vay<br />
ddc tinh ciia cac phan doan nay la do cac neurotoxin gay ra. Cdn cac phan doan khdng doe 2,<br />
3, 4 chira chii yeu cac protein nam trong cac viing cd khdi Iugng phan tir 30-70 kDa va 10-14<br />
<br />
<br />
<br />
<br />
Hinh 2: Kit qud dien di ciia mdt sd phdn dogn tdch ra tu nge bd cgp qua cdt<br />
Trong dd: 1-phan doan 2; 2-marker; 3-pban doan 3; 4-phan doan 4; 5-phan doan 5; 6-phan<br />
doan 6; 7-pban doan 7.<br />
Kda. Phan doan 5 chiia chu yeu cac peptit cd khdi Iugng phan tir ndm chii yeu trong vimg 3-<br />
14 kDa. Trong dd cac peptit cd khdi lugng phan tir 3-8 kDa gidng khdi lugng phan tir ciia cac<br />
neurotoxin, nhung chiing khdng cd ddc tinh, ban chdt cac peptit nay kbac vdi neurotoxin noc<br />
bd cap.<br />
154<br />
Hoi nghi Khoa hqc ky niem 35 ndm Vien Khoa hqc vd Cong nghe Viet Nam - Hd Noi 10/2010<br />
<br />
<br />
3. LDso duong tiim duoi da<br />
Cac bieu hien ciia chuot trong vdng 24 gid khi tiem dudi da noc bd cap: sau \"ai phiit,<br />
chuot bat dau gai mdm lien tuc, tang tiet nude hot rdt nhilu, tang tilt md hdi, co giat nhe cac<br />
CO va chuot chit trong vdng 1,5-3 gid.<br />
Chuot khdng chet dugc theo ddi tiep trong 14 ngay va giai phau quan sat dai thi<br />
khdng quan sat thdy cac tdn thuong {bdng 2)<br />
Bdng 2. Kit qud sd chugt chit trong cdc Id sau 24 gl& dim du&l da nge bd cgp<br />
Theo phuong phdp Miller-Tainter<br />
<br />
Lieu (mg/Kg) Chuot/lo Chuot chet/16 % chuot chet<br />
<br />
110 (LDo) 6 0 0<br />
140 9 2 22,22<br />
Noc bo cap Tay<br />
170 15 8 53,33<br />
Ninh<br />
{H. Laoticus) 200 15 11 73,33<br />
230 9 7 77,77<br />
260 (LDioo) 6 6 100<br />
Ve dudng bieu dien lilu theo ti le % chit theo lilu tren gidy logarit. Tir dd thi, ta suy<br />
ra cac gia tri: m; Probit 6,0 (84% chit); Probit 4,0 (16% chit); 2S; N'; E. Suy ra LD5o= 170 ±<br />
9,95 (mg/Kg)<br />
Vay lilu LD50 ciia noc bd cap den dudng tiem dudi da tren chuot nhdt trdng DDY, 18<br />
- 25 g la 170 ± 9,95 mg/Kg theo phuong phap Miller - Tainter.<br />
Theo phuong phdp Karber-Behrens.<br />
LD50 ciia noc bd cap den khi tiem dudi da tren chuot nhdt trdng DDY, 18 - 25 g la 167<br />
mg/Kg. Theo phan loai ciia Ernest Hodgson, Patricia E. levi (2000) [5], noc ciia loai bd cap<br />
nay dugc xep vao nhdm doe loai 111- ddc vua phai.<br />
4. Kit qud tim nghiem tdc dgng gidm dau dau trung itong theo phuong phdp<br />
nhung dudi chuot<br />
Tat ca cac chuot d cac Id trong qua trinh thir nghiem diu khoe manh, khdng cd bilu<br />
hien bdt thudng. Chuot dugc nudi giir 03 ngay sau khi thir nghiem diu song khde manh. Tiln<br />
hanh thir nghiem tac dung giam dau trung uongtheo md hinh nhiing dudi chuot cho kit qua<br />
nhu sau (Hinh 3)<br />
<br />
10.00<br />
<br />
io 8.00<br />
3<br />
6.00<br />
c<br />
<br />
<br />
<br />
<br />
M Jffl.JI<br />
-3<br />
C 4.00<br />
C<br />
ra<br />
•& 2.00<br />
-•5<br />
5 0.00<br />
30 phut 60 phut 90 phut<br />
thcri d i l m nhung duoi (phut)<br />
<br />
n Chirng • Morphin 2,5 D Den li§u 8,5 D Den li^u 17<br />
Hinh 3. Th&i gian chugt cd phan irng gigt mgnh dudi ra khdi nu&c ndng & cdc id<br />
chirng morphin liiu 2,5 mg/Kg vd nge bd cgp liiu 8,5 vd 17 mg/Kg tai cdc ih&i diem thu<br />
nghiem 30. 60 va 90 phut sau khi chugt dugc dim thudc. Sd liiu biiu diin du&i dang Irung<br />
binh ± SEM. **P< 0.01 so v&i Id chirng -"P
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