Nghien CLFU - Ky thuat
Doc tinh cap - ban truong dien va tac dong
giam dau, khang viem cua noe bo cap den An Giang {Heterometrus laotieus)
V6 Phung Nguyen', huu Hoang Le Giang', Hoang Ngoc Anh 'Truang dgi hoc YDirac TP.HCM
^Vien Hoa hoc img dung TP.HCM
D a t v a n d e
Tir lau, bd cap da dygc sir dung rdng rai va la mdt v| thudc quy trong cac bai thudc co truyin cua nhiiu nyde Viet Nam, Trung Quoc, Trieu Tien...de trj ddng kinh d tre em, udn van, ban than b i t toai, thien d i u thing, trang nhac, quai bj... dydi dang thudc bpt hoac lam vien udng.
Tuy nhien, thanh phin chinh ed tae dung la noe bd cap v i n chya dygc nghien eiru nhiiu. G i n day, npc bd cap da thu hiit dygc sy quan tam cua cac nha khoa hpc de tim hieu ve thanh phan, tinh c h i t ly hda, cae tae dung dygc ly eung nhy ddc tinh cua chiing. Nhieu cdng trinh nghien ciru da d y g c cdng bo cac tac dung irng dung cua npc bd cap trong y dygc nhy: kaliotoxin, mdt c h i t cd tac ddng (yc che kenh Kali cd trong npc cua nhiiu loai bd cap da dygc nghien ciru va cho thiy cd t^c ddng khang viem va ngan m i t xyong '"*', chlorotoxin tir npc bd cap vang Israel cd tac dung hd trg trong dieu trj ung thy nao '^'...va g i n day npc bd cap den thu mua d Tay Ninh eung da d y g c cdng bo cd tac dung giam dau, khang viem '^'. Trong nghien ciru nay, ehiing tdi t i i n hanh khao sat ddc tinh c i p , ban trydng d i i n va cac tac ddng giam dau, khang viem cua npc bd cap Heterometrus laotieus thd thu mua d An Giang.
N g u y e n lieu v a phu'O'ng p h a p Noe bo cap
Bd cap trydng thanh dai 8-12,5 cm, mua d An Giang, dygc kieh thich bing may tao xung dien tai ddt thir 5 hoac 6 de thu npc, sau dd lam khd trong binh hut i m cd chira CaCIa khan eho d i n khi khoi lygng khdng doi va bao quan d -20 °C. Npc dygc hda trong nyde muoi sinh ly thanh dung dieh de khao s^t dde tinh vS tac dung dygc ly.
Dong vat thiF nghiem
Chudt nhit tring giong ddY, ca hai gidi, trpng lygng 18-25 g do vien Pasteur thanh phd Ho Chi Minh eung eip. Chudt mua v i dygc de on djnh 2 ngay trong phdng thi nghiem dygc ly dai hpc Y Dygc TP.HCM trydc khi t i i n hanh thy nghiem.
Thdi gian thCr nghiem tir 8-15 gid.
Nghien cii'u doc tinh
Nghien ciru doc tfnh cap dwang tiem dwai da tren chuot nhat trang '^'
Khao sat ddc tinh e i p d y g c thyc hien theo hydng d i n cua t^c gia Dd Trung Dam, tyong tir nhy da cdng bo d eae bai bao trydc '^l
Nghien ciru so khdi: Khdi d i u tir lieu cao nhit cd the qua dygc kim cho udng. Xac djnh liiu LDo (lieu toi da khdng gay chit) va lieu LD100 (liiu toi thieu gay ehit 100%).
Nghien ciru xic djnh: Chudt dygc chia lam 6 Id, mdi Id it nhit 6 eon, cho su- dung thuoc d cac liiu trong khoang LDQ va LD100, chia theo c i p so.
& nhu-ng liiu g i n LD50 tang sd lygng chudt de sir do lydng dygc chinh xac hon. Quan s^t chudt trong 24 gid sau khi diing thudc, ghi nhan eae phan irng xay ra tren chudt va sd lygng chudt song, c h i t d mdi Id. Theo ddi chudt cdn sdng sau 14 ngay ke tir ngay dung thudc. Giai phiu dai the nhO-ng chudt edn sdng va nhlj-ng chudt ehit sau khi cho uong. LD50 d y g c tinh theo phyong phap Miller - Tainter va Karber - Behrens.
Nghien cCru dgc tinh ban trwang dien dwang tiem dwai da tren chugt nhat tring
Chudt dygc chia n g i u nhien thanh 2 Id:
-Ld chirng: 20 con -Ld thy: 30 con
Chudt dygc tiem dydi da dung djch nyde muoi sinh iy (Id chirng) va dung dich npc bd cap
TAP CHI DUQC HQC - 8/2009 (SO 400 NAM 49) 13
9,5 mg/kg (Id thCr), vdi lygng 0,1 mg/ 10 g the trpng/ ngay.
Ca 2 Id chirng va thu- d y g c nudi trong ciing mdt d i i u kien va theo ddi trpng lygng cu 5 ngay mdt l i n . Thy nghiem dygc tien hanh trong 1 thang. Sau thy nghiem chudt dygc l i y mau de xet nghiem v i huyit hpc, hda sinh va sinh thiit gan than.
Nghien cii'u tac dung du'ge ly
Liiu nghien eiru tac dung d y g c ly d y g c suy ra bing each l i y 1/20- 1/10 gia trj LD50. Chudt dygc chia thanh 4 Id, mdi Id la 8 - 10 chudt. (2 Id thCr d hai lieu 1/20 va 1/10 cua gia tri LD50, 1 Id chit-'ng va 1 Id ddi chirng), rieng nghien ciru tac dung khang viem cdn them Id tring (tiem dydi da dung mdi pha thudc ddi ehirng)
Nghien CIFU tac dung giam dau trung u'O'ng theo phu'O'ng phap nhung duoi chuot '^'
Chudt dygc cd dinh vao ldng vdi dudi d y g c tha tu- do ra ngoai. Nhiing ngap dudi chudt khdng qua 5 cm trong binh nyde d nhiet dp on dinh 50
± 0,5 °C. Thdi gian giat manh dudi chudt tinh tir liic nhiing dudi vao nyde den khi chudt giat manh dudi ra khoi nyde, d y g c ghi nhan trydc khi cho dung thude va vao cac thdi diem 30, 60, 90, 120 phut sau khi eho thudc. Lau khd dudi chudt sau mdi l i n do, neu chupt khdng phan irng sau 10 giay thi nhie chudt ra de tranh bdng dudi cho chudt. So sanh t i i m thdi giat manh dudi cua chudt giu-a cae Id vdi nhau.
Nghien CCFU tac dung giam dau ngoai bien theo mo hinh gay dau quan '^^
Sau khi cho chudt sCr dung thuoc nghien eiru 30 phiit, tiem phuc mac l i n lygt tirng chudt vdi 10 mL/kg acid acetic 0,7%. Sd l i n dau quan cua chudt dygc quan sat va ghi nhan trong 5 phiit d cac thdi diem 5-10, 20-25 va 35-40 phiit sau khi tiem acid acetic. Chudt d y g c eho la dau quan khi dau quan bung va dudi it nhit mdt chan sau. So sanh sd l i n dau quan cua chudt giCra cae Id tai cac thdi diem vdi nhau.
Nghien ciPU tac dong khang viem theo mo hinh gay phu ban chan chuot b l n g carrageenin '^'
Carrageenin dygc cung c i p bdi Sigma Aldrich, dygc diing lam tae nhan gay viem. Oo dp phil ban chan chudt bing Plethysmometer model 7140, hang Ugo Basile.
Chudt dygc gay viem bing each tiem vao gan ban chan sau 0,025 mL dung djch carrageenin
1% pha trong dung djch sinh ly. Oo the tich ban chan chudt 3 gid sau khi gay tiem. Cae chudt cd the tieh chan syng phii tren 50% so vdi binh thydng dygc lya ehpn eho nghien ciru va chia ngiu nhien thanh cac Id va l i n lygt tiem dydi da eae dung dich: Ld chirng: dung djch sinh ly 0,09%; Ld ddi chirng: dung djch ketoprofen (Profenid®), 5 mg/kg/ benzyl alcohol 2,5 %; Ld tring: dung dich benzyl alcohol 2,5 %; Ld thy thuoc dung djch npc bd cap d hai liiu khac nhau.
Theo ddi the tich syng phii cua ban chan chudt sau 3 gid gay viem va sau khi tiem thudc 30 phiit mdi ngay vao mdt gid n h i t dinh trong 6 ngay lien tiip. Op tang the tich chan cua tirng chudt cdn gpi la dp syng phii, d y g c tinh theo cdng thirc:
Vt-V
AV% = ^xlOO
^°
VQ: The tich chan chudt trydc khi gay viem (do 1/100 mL)
V t : The tich chan chudt sau khi gay viem (do 1/100 mL)
Thdng ke phan tich
DO- lieu dygc trinh bay dydi dang sd trung binh ± SE. Sy khac biet giO'a cac nhdm dygc phan tich bing phyong phap Kruskal-Wallis sau dd la Mann-Whitney test vdi p h i n m i m vi tinh Minitab 14.0. P < 0,05 d y g c cho la cd y nghla thdng ke.
K i t qua
LD50 du'o'ng tiem du'd'i da
Cac bieu hien eua chudt trong vdng 24 gid sau khi tiem dydi da npc bd cap: sau vai phut, chudt b i t d i u gai mdm lien tuc, tang tiet nyde bpt r i t nhiiu, tang t i i t md hdi, co giat nhe cac co va chudt ehit trong vdng 1 , 5 - 3 gid. Chupt khdng chit d y g c theo ddi t i i p trong 14 ngay va giai phiu quan sat dai the khdng quan sat thiy cac ton thyong.
Liiu LD50 eua npc bd cap den dydng tiem dydi da tren chudt n h i t tring ddY, 18 - 25 g la 190 ± 1,74 mg/kg tinh theo phyong phap Miller - Tainter va 188.68 mg/kg tinh theo phyong phap Karber- Behrens.
Doc tinh ban tru'dng d i i n
v i the trang ehung: trong sudt qua trinh thy nghiem, khdng cd chudt chit. Trong nghien ciru nay, do d i n ngay thir 18 mdt so chupt Id thy b i t
14 TAP CHI DUQC HOC - 8/2009 (SO 400 NAM 49)
Nghien CLFU - Ky thuat
dau bj hoai tCr va bong trdc viing da lyng b| tiem nen thy nghiem dygc k i t thiic sau 30 ngay. T h i trpng trung binh chudt giira 2 Id d y g c so sanh tai cac khoang thdi diem cce 5 ngay mdt l i n . Chudt d 2 Id tang trydng binh thydng va khac nhau khdng cd y nghTa trong sudt 30 ngay thy nghiem.
Hinh 1: Sw thay ddi thi trong chudt qua cac ngay Chudt dygc mo de xet nghiem vao ngay thir Bang 1 : Kit qua xet nghiem huyit hoc so sanh
31 sau khi gay me b i n g da CO2 khd.
vi huyit hoc: Mdt sd thanh phin trong edng thirc mau ed thay doi, khac biet gitra Id chirng va Id thu- nghiem tuy nhien van n i m trong gidi han binh thydng sau 30 ngay thu- nghiem. Lygng hdng eau va Hemoglobin giam khoang 10% so vdi Id chirng (p<0,05). Sd lygng bach cau eua Id thy giam ed y nghTa so vdi Id chirng vdi ty le bach c i u trung tinh tang va te bao lympho giam ed y nghTa so vdi chirng cd the do nhiim triing do hoai tu- viing da tiem cua Id thy. Cac loai bach c i u khac nhy bach c i u don nhan to, bach c i u ya eosin, bach e i u ya bazo thay doi khdng dat y nghTa thing ke. Sd lygng tieu c i u cua Id thy cung tang nhyng chya khac dat y nghTa thing ke.
giwa Id thir va Id chirng sau 1 thing thir nghiem
^ ^ ^ C h / so Lo ^ ^ ^
Chirng Thir
SL hong cau (xloVmm^) 9,96 ± 0,24 8,99 ± 0,25 *
Hemoglobin (g/di) 15,75 ±0,7 14,37 ± 0 , 5 5 *
SL bach cau
(xl6^/mm^)
13,67 + 2,43 10,41 ± 2 , 5 *
SLtieu cau (xloVmm^)
620 ± 295 950,1 ±182,2 (*) p<0,05, khac cd y nghTa thdng ke so vol Id chirng.
Ve chirc nang gan va than: sau 30 ngay sCr Creatinin) cua chudt d hai Id chirng va su- dung dung npc bd cap tiem dydi da, cac chi sd hda npc khac nhau khdng cd y nghTa.
sinh v i gan (SGPT, SGOT, Bilirubin) than (BUN,
Bang 2: Kit qua xet nghiem hda sinh so sinh giCea Id thir vi Id chirng sau mot thing thir nghiem Chi so
Lo \ SGOT (U/L)
Sinh hoa gan - mat Sinh hoa than
SGPT Bilirubin (T) Bilirubin (D) (U/L) (mg/dl) (mg/dl)
Bilirubin Creatinin (I) BUN (mg/dl) huyet thanh
(mg/dl) (mg/dl) Chirng
(n=9) Thu- (n=8)
159,9 + 38 165 ±35,6
62,4 ± 14,7 69,22±15,3
1
0,2286 ±0,05 0,2333 ±0,05
0,1143 ±0,04 0,1333±0,05
0,1 0,1
20,29 ±3,99 20,67 ±3,81
0,59 ±0,04 0,62 ± 0,04 v i giai phiu vi thi: khdng phat hien sy bit bao gan binh thydng vdi khoang cu-a tham nhap thydng tren than cua chudt d ca hai Id ehirng va thy.
Tren gan, 1/10 m i u gan cua Id thy quan sat cd t i Tac dung giam dau
Mo hinh nhung duoi chugt:
nhieu lympho bao va 1/10 mau gan cd te bao gan ed nhan va bao tyong phdng to, nhiiu khdng bao.
J i i
I 3 0 phut S a u 6 0 phut S a u 9 0 phL S a u 1 2 0 p h u t
• C h u n g ^ M o r p h i n [Z3 L i e u 19 m g / k g • L i ^ u 9.5 m g / k g
Hinh 1. Tiim thoi giat manh dudi trung binh ciia cac Id vio cic thoi diim
*p<0,01, khic cd y nghTa thdng ke so vdi Id chimg; # p<0,01, Mp<0,001, khic cd y nghTa so voi Id morphin
TAP CHi DUQC HQC - 8/2009 (SO 400 NAM 49) 15
Trydc thy nghiem: tiem thdi giat manh dudi cua chupt d tat ca cac Id khac nhau khdng cd y nghTa thing ke.
Ld morphin: co tac dung giam dau, lam tang tiem thdi cd phan u'ng giat dudi cd y nghla so vdi Id chirng tir phiit 30 va keo dai d i n 120 phiit. Ld liiu 19 mg/kg va 9,5 mg/kg: d i u lam tang t i i m thdi giat manh dudi cd y nghTa thing ke so vdi Id chirng tir phiit 30 va keo dai d i n 90 phiit, nhyng y i u hon morphin. T i i m thdi giat manh dudi chupt cua 2 liiu khac nhau khdng cd y nghTa.
Mo hlnh gay dau quan:
Ld aspirin: cd tac dung giam dau, lam giam sd l i n dau quan tai ca 3 khoang thdi gian 5-10, 20- 25, 35-40 phiit. Npc bd cap d ca 2 lieu d i u cd tac dung giam sd l i n dau quan nhy nhau, lam giam so l i n dau quan d ca 3 khoang thdi gian cd y nghTa so vdi Id chirng va Id aspirin liiu 50 mg/kg.
Tac dung khang viem
J
i
120
— 100 .T 80
- 60
a 20 0
3 giff Ngay 1 Ngay 2 Ngay 3 Ngay 4 Ngay 5 Ngay 6
-Chuiig —•—Trang -l.icu !9iiigkg —B—Lieu 9."i mg.'kg
-Kelt) pro ten
Hinh 2. Sd lin dau quan trung binh ciia cac Id vao cic thai diim.
* p<0,001, khac cd y nghTa thdng ke so vdi Id chirng; # p<0,001, khac co y nghTa so vol Id aspirin
Bang 3: Do phii ban chan chudt viem (%,) tai cac thdi diim
Hinh 3: Phan tram do swng phii ban chan chudt sau 3 gio tiem carrageenin (ngay 0) va 30 phiit sau tiem thudc mdl ngay (trong 6 ngay).
L6
Chirng Trang Ketoprofen
2,5 mg/kg Lieu 19 mg/kg
Lieu 9,5 mg/kg
3 giiy 86,38
± 19,1 89,41
±6,73 88,6
±19,9 87.52
± 14,99 80,94
± 16,8
Ngay 1 80,87
±14,92 73,83
±5,23 51,55 + 15**##
53,18 ± 3,54**
47,72 ± 8,21 ***
Ngay 2 79,74
± 14,95 63,28
± 7 , 5 1 * 46,94 ± 13,69***#
40,43 ± 15,38**
37,35
± 5 , 0 3 * *
Ngay 3 76,54 + 13,02
59,18
±10,77*
38.22 ± 10,9***##
37,09 ± 9,73 ***
38.29 ± 20,33**
Ngay 4 63,12
± 12,58 55,46
±7,63 36,04 ± 11,5***##
33,23 ± 10,67**
35,24
± 13,95**
Ngay 5 58,91
±11,24 53,85
± 12,83 30,39 + 12,79***##
13,64
± 3,6 '*¥
23,73 ± 12,95***
Ngay 6 47,61
±8,28 40,99
±2,55 24,58 ± 17,47**#
8,53
± 5 , 5 3 * * 9,45 ± 6,58 ***
(*), p<0,05: (**), p<0,01; (*"*), p<0,001, khac cd y nghTa thdng ke so voi Id chirng.
(It), p<0,05; (##). p<0,01;, khac cd y nghTa thing ke so vdi Id tring (benzyl alcohol).
(¥), p<0,05. khac cd y nghTa thdng ke so vdi Id ketoprofen Ld dung mdi benzyl alcohol: dp phii ban chan
ctiudt giam khac nhau khdng cd y nghTa thing ke so vdi Id chirng qua cac ngay 1, 4, 5, 6. Vao ngay thir 2 va 3, lam giam dp syng phii khac nhau cd y nghTa thing ke so vdi Id chirng. Benzyl alcohol lam giam dp syng phu khac nhau khdng cd y nghTa thong ke so vdi Id ketoprofen qua cac ngay.
Ld ketoprofen: ketoprofen lam giam phii ban chan chudt cd y nghTa thing ke so vdi Id chirng va so vdi Id benzyl alcohol tir ngay 1 d i n ngay 6.
Vay benzyl alcohol diing de pha ketoprofen khdng cd tac ddng gia tang hay giam bdt tac ddng khang viem cua ketoprofen.
Npc bo cap liiu 19 mg/kg lam giam dp syng phii ban chan chudt cd y nghTa thing ke so vdi Id chirng tir ngay 1 d i n ngay 6, va so vdi Id ketoprofen d ngay 5. Vao ngay thir 6 lam giam dp syng phii so vdi Id ketoprofen nhyng chya dat y nghTa thong ke.
16 TAP CHI DUQC HQC - 8/2009 (SO 400 NAM 49)
Nghien CLFU - Ky thuat
Npc bd cap liiu 9,5 mg/kg lam giam dd syng phil ban chan chudt cd y nghTa so vdi Id chirng tir ngay 1 den ngay 6. Od giam syng phii khae nhau khdng cd y nghTa so vdi Id ketoprofen va Id npc liiu 19 mg/kg.
Tac ddng lam giam dp syng phii ban chan chudt cua npc bd cap khac nhau khdng cd y nghTa giira 2 liiu.
K§t luan
Npc bd cap cd ddc tinh c i p LD50 la 190 ± 1,74 mg/kg khi tiem dydi da d chudt nhit tring ddY va dygc x i p vao nhdm ddc loai III '^', ddc vira phai.
Npc bd cap den Heterometrus laotieus b i t tai An Giang, tiem dydi da liiu 9,5 mg/kg va 19 mg/kg cd tac dung giam dau len t h i n kinh trung yong tren md hinh nhiing dudi chudt y i u hon morphin tiem dydi da liiu 5 mg/kg va tac dung giam dau ngoai bien tren md hinh dau quan gay ra bdi acid acetic tot hon aspirin udng lieu 50 mg/kg. Tac ddng khang viem tyong dyong vdi ketoprofene tiem dydi da lieu 2,5 mg/kg.
Npc bd cap tiem hang ngay dydi da vdi liiu 0,1 mg/10g the trpng trong /ngay trong 30 ngay gay hoai tir da nhyng chya bieu hien ddc tinh tren gan, than, va huyit hpc trong mdt thang thir nghiem.
Summary
Scorpions have been used for a long time in traditional medicines of many eastern countries, and demonstrated many medicinal effects, such as anticonvulsion, antiinflammatory, analgesic activities ... The venom, as the main bioactive ingredient of the insect, has been widely investigated in the recent 3 decades and demonstrated many pharmacological significances. In this study, the venom of the black scorpion from An Giang (Heterometrus laotieus) were investigated for acute and subacute toxicity, analgesic, antiinflammatory, and anticonvulsive activities in mice by subcutaneous administration. The animals were observed for 24hs after the venom injection to evaluate the lethal dose. The anti-ociceptive effect of the venom was investigated by tail immersion and acetic acid-induced writhing tests.
The in vitro anti-inftammatory activity was investigated by carrageenin-induced hind paw edema in mice. The subchronic toxicity of venom was also investigated in mice.
The LD50 of the tested black scorpion venom was 190 ± 1.74 mg/kg B.W. mouse (s.c). Toxicity of the black scorpion venom is classified into the third class. The black scorpion venom showed both central and peripheral analgesic effects by the acetic acid induced writhing and tail Immersion tests, as well as the anti-inflammatory effect by carrageenin-induced inflammation test at the dosages of 9.5 or 19 mg/Kg B.W. mouse (s.c). No noticeable signs of subacute toxicity after a month of subcutaneous administration.
Keywords: analgesic activity; anti- inftammatory activity; Heterometrus laotieus;
LDso; scorpion; venom.
Tai lieu tham khao
1. D6 Trung Dam (1996), Phyong phap xac dinh doc tinh c§p cua thu6c, NXB Y hoc. Ha Noi.
2. Ernest Hodgson, Patricia E. levi (2000), A textbook of mordern toxicology, pp. 285-296.
3. Koster R, Anderson M, de Beer EJ (1959) Acetic acid for analgesic screening, Fed Proc 18:412
4. Martin A. Taubman, Paloma Valverde, Xiaozhe Han, Toshihisa Kaw/ai (2005), "Immune Response: The Key to Bone Resorption in Periodontal Disease", J.
Pehodontol, 76(11), 2033-2041.
5. Nguy§n Thi Phyang Khue, V5 Phiing Nguyen, Hoang Ngpc Anh (2008), "Nghien ciru dpc tinh cip, tac dung giam dau, khang viem cua npc bo cap den Tay Ninh". Tap chi Ducrc hoc, 389, 03/2008,
6. Soroceanu Liliana, Gillespie Yancey, Kharazaelil M. B., Sothelmer Harald (1998), "Use of Chlorotoxin for targeting of primary brain tumors". Cancer Res..
58(21), 4871-4879.
7. Taber Rl, Greenhouse DD, Rendell JK, Irwin S (1969) Agonist and antagonist interactions of opioids on acetic acid-induced abdominal stretching in mice. J Pharm Exp Ther 169:29-38
8. Winter, E. Risley and G: Nuss (1962),
"Carrageennan- induced edema in hind paw/ of the rat as an assay for anti-inflammatory drugs", Proceedings Society of Experimental Biological Medicine, 111, pp.
544-547.
TAP CHi DU'QC HQC - 8/2009 (SO 400 NAM 49) 17
